Angela Ianaro

Modulation of acute inflammation by endogenous nitric oxide

The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(L-NAME) and NG-monomethyl-L-arginine (L-NMMA)) as well as L- or D-arginine. The effect of test compounds was studied on the carrageenin-induced increase in vascular permeability in rat skin and in dextran- and carrageenin-induced paw oedema. Both L-NAME and L-NMMA dose dependently inhibited the increase in vascular permeability and oedema formation. L- but not D-arginine increased these inflammatory responses and reversed the inhibitory effects of L-NAME and L-NMMA. In dexamethasone-treated rats L-arginine enhanced the dextran-induced oedema and the early phase of carrageenin-induced oedema but did not modify the inhibition by dexamethasone of the late phase of carrageenin-induced oedema. These results suggest that endogenous NO is released at the site of acute inflammation and modulates oedema formation. Depending on the time course or on the type of inflammation, NO may be predominantly generated by the constitutive or by the inducible NO synthase.

Phytochemical compounds involved in the anti-inflammatory effect of propolis extract

Two ethanolic propolis extracts (EPE) with and without the caffeic acid phenethyl ester (CAPE), CAPE and galangin (major components of propolis) were investigated for anti-inflammatory activity in rats using carrageenin foot oedema, carrageenin pleurisy and adjuvant arthritis. In our experiments, EPE with CAPE and CAPE alone significantly inhibited carrageenin oedema, carrageenin pleurisy and adjuvant arthritis. In contrast EPE without CAPE and galangin did not exhibit anti-inflammatory effects in acute and chronic inflammation. These results suggest that the anti-inflammatory activity of propolis is due to CAPE.

Modulation by nitric oxide of prostaglandin biosynthesis in the rat.

1 Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)‐induced paw oedema and measuring both the foot volume and the amount of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2 Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor l‐NG‐nitro arginine methyl ester (l‐NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 μmol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 μmol/paw) and L‐arginine (15 μmol/paw). 3 The NO‐donors (10 μmol/paw) 3‐morpholino‐sydnonimine‐hydrochloride (SIN‐1), S‐nitroso‐N‐acetyl‐d, l‐penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4 SIN‐1 (2.5, 5 and 10 μmol/paw) produced a significant dose‐dependent increase of the oedema induced by AA which was correlated with increased amounts of 6‐keto‐PGF1α in the fluid recovered from inflamed paws. 5 Both oedema and prostaglandin biosynthesis induced by the combination AA + SIN‐1 were greatly suppressed by either Hb (30 μmol/paw) or indomethacin (3 μmol/paw or 5 mg kg−1 s.c.) but unaffected by Mb (3 μmol/paw). 6 In LPS‐treated rats (6 mg kg−1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by l‐NAME or Hb, unaffected by Mb and increased by l‐arginine. 7 These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3′:5′‐cyclic monophosphate (cyclic GMP)‐independent mechanism and suggest that the interaction between NO synthase and cyclo‐oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

Rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ), reduce ischaemia/reperfusion injury of the gut

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2), are two PPAR‐γ ligands, which modulate the transcription of target genes. The aim of this study was to investigate the effect of rosiglitazone and 15d‐PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β levels and marked injury to the distal ileum. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti‐intercellular adhesion molecule (ICAM‐1) antibody resulted in diffuse staining. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR‐γ agonists (rosiglitazone (0.3 mg kg−1 i.v.) and 15d‐PGJ2 (0.3 mg kg−1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF‐α and IL‐1β) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d‐PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM‐1 during reperfusion. In order to elucidate whether the protective effects of rosiglitazone and 15d‐PGJ2 are related to the activation of the PPAR‐γ receptor, we also investigated the effect of a PPAR‐γ antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d‐PGJ2. BADGE (1 mg kg−1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d‐PGJ2) significantly antagonised the effect of the two PPAR‐γ agonists and thus abolished the protective effect against gut I/R. These results demonstrate that the two PPAR‐γ agonists, rosiglitazone and 15d‐PGJ2, significantly reduce I/R injury of the intestine.

The cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 attenuates the development of colon injury caused by dinitrobenzene sulphonic acid in the rat

Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and increased expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM‐1) in the colon. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15‐deoxy‐Δ12,14‐PGJ2 (15d‐ PGJ2) functions as an early anti‐inflammatory signal. The aim of the present paper is to investigate the effects of 15d‐PGJ2 in rats subjected to experimental colitis. Colitis was induced in rats by intra‐colonic instillation of dinitrobenzene sulphonic acid (DNBS). 15d‐PGJ2 was administered daily as intraperitoneal injection (20 or 40 μg kg−1). On day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. 15d‐PGJ2 significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. 15d‐PGJ2 also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde (MDA) and (iv) of the pro‐inflammatory cytokines tumour necrosis factor‐alpha (TNF‐α) and interleukin‐1β (IL‐1β). Furthermore, 15d‐PGJ2 reduced the increase in immunohistochemical staining for (i) inducible nitric oxide synthase (iNOS), (ii) nitrotyrosine and (iii) poly (ADP‐ribose) polymerase (PARP), as well as (iv) the increased expression of ICAM‐1 caused by DNBS in the colon. Electrophoresis mobility shift assay (EMSA) of inflamed colon revealed that 15d‐ PGJ2 also caused a substantial reduction of the activation of nuclear factor‐kappaB (NF‐κB). Furthermore, 15d‐PGJ2 stimulates the activation of heat shock protein 72 (hsp72) in the inflamed colon, as assessed by Western blot analysis. In conclusion, 15d‐PGJ2 reduces the development of experimental colitis.

The IκB Kinase Is a Key Factor in Triggering Influenza A Virus-induced Inflammatory Cytokine Production in Airway Epithelial Cells

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IκB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-κB (NF-κB), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-κB pathway by influenza virus infection, NF-κB was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-κB was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenza-induced inflammation and airway pathology. We also report that 9-deoxy-Δ9,Δ12-13,14-dihydro-prostaglandin D2, a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.

Glycolipids from sponges. IV. Immunomodulating glycosyl ceramides from the marine sponge agelas dispar.

The GSL composition of the marine sponge Agelas dispar was investigated. In addition to four GSLs previously isolated from Agelas clathrodes (2 and 4), Agelas conifera (3), and Agelas longissima (5), the novel triglycosylceramide 1a was isolated as a major component of the GSL mixture. All the isolated GSLs were tested using the MLR assay, and only some of them were shown to be immunoactivating agents, suggesting a possible structure-activity relationship.

Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.

In this study, using rat carrageenin‐induced pleurisy, we found that treatment of rats with either indomethacin or NS‐398 suppressed the pleurisy at 2 h but significantly exacerbated this reaction at 48 h. Exacerbated inflammation was associated with reduced prostaglandin D2 levels, decreased heat shock factor 1 (HSF1) activation, reduced hsp72 expression and increased activation of nuclear factor κB (NF‐κB). Replacement of cyclopentenone prostaglandins by treating rats with either prostaglandin J2 or prostaglandin D2 reversed the exacerbating effects of cyclooxygenase inhibitors leading to the resolution of the reaction. In conclusion, we demonstrate that cyclopentenone prostaglandins may act as anti‐inflammatory mediators by inducing in inflammatory cells HSF1‐dependent hsp72 expression and NF‐κB inhibition, two crucial events for the remission of inflammation.

Metabolites from the sponge Plakortis simplex. Determination of absolute stereochemistry of plakortin. Isolation and stereostructure of three plakortin related compounds

Abstract The polyketides dihydroplakortin ( 3 ), and the dodecanoic acid derivatives 4 and 5 , were isolated from the Caribbean marine sponge Plakortis simplex , and their structures fully characterized by spectroscopic and chemical means. The absolute stereochemistries of the known plakortin ( 1 ) and of compounds 3–5 were determined by applying Moshers and Kusumis methods on opportune degradation products. The isolated compounds exhibited cytotoxic activity tested in vitro on WEHI 164, murine fibrosarcoma cell line.

Relationship between nitric oxide and prostaglandins in carrageenin pleurisy

The correlation between endogenous nitric oxide (NO) generation and prostaglandin biosynthesis was studied in rat carrageenin pleurisy induced by the injection of 0.2 mL of 1% lambda-carrageenin into the pleural cavity. The pleural exudate was collected at 4 hr and the amounts of NO2- + NO3- (NOx) and prostaglandin E2 (PGE2) measured. The NOx present in the inflammatory exudate was determined by measuring the NO2- with the Griess reaction, after the reduction of NO3- to NO2- using acid-washed cadmium powder. PGE2 was measured by radioimmunoassay. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1-3-10 mg/kg subcutaneously) reduced NOx by 20 +/- 7%, 41 +/- 6% and 55 +/- 9% (P < 0.01) and PGE2 by 9 +/- 6%, 41 +/- 11% and 74 +/- 9% (P < 0.001). Conversely, L-arginine (300 mg/kg SC) increasedNOx by 39 +/- 7% (P < 0.01) and PGE2 by 78 +/- 6% (P < 0.001). The NO scavenger haemoglobin (Hb), coinjected into the pleural cavity (3 mg/site) with carrageenin, produced a parallel inhibition of NOx (65 +/- 16%, P < 0.001) and PGE2 (71 +/- 18%, P < 0.001). The soluble guanylate cyclase inhibitor methylene blue (Mb; 2 mg/site) had no effect. Moreover haemoglobin, but not methylene blue, was able to significantly suppress the L-arginine-induced increase of both NOx and PGE2. In each pleural exudate, independently from the animal treatment, the amount of NOx was highly correlated to the amount of PGE2 (r = 0.93, P < 0.001). These results suggest that in rat carrageenin pleurisy the modulation of the L-arginine:NO pathway results in a parallel modulation of prostaglandin biosynthesis. The interaction between cyclooxygenase and the NO pathway may represent an important mechanism for the modulation of the inflammatory response.