Barbara Federici

Galectin-1 suppresses experimental colitis in mice

BACKGROUND & AIMS Uncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacologic strategies directed to restore the normal responsiveness of the immune system by deleting inappropriately activated T cells could be efficacious in the treatment of these pathologic conditions. Galectin-1 is an endogenous lectin expressed in lymphoid organs that plays a role in the maintenance of central and peripheral tolerance. The aim of the present study was to evaluate the therapeutic effects of galectin-1 on T-helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. METHODS Cells and tissues from mice with TNBS colitis receiving treatment with several doses of human recombinant galectin-1 (hrGAL-1) were analyzed for morphology, cytokine production, and apoptosis. RESULTS Prophylactic and therapeutic administration of rhGAL-1 resulted in a striking improvement in the clinical and histopathologic aspects of the disease. hrGAL-1 reduced the number of hapten-activated spleen T cells, decreased inflammatory cytokine production, and profoundly reduced the ability of lamina propria T cells to produce IFN gamma in vitro. Moreover, hrGAL-1 led to the appearance of apoptotic mononuclear cells in colon tissue when administered in vivo and induced selective apoptosis of TNBS-activated lamina propria T cells in vitro. CONCLUSION Collectively, these data show that hrGAL-1 exerts protective and immunomodulatory activity in TNBS-induced colitis and it might be effective in the treatment of inflammatory bowel diseases.

NO-Aspirin Protects From T Cell-Mediated Liver Injury by Inhibiting Caspase-Dependent Processing of Th1-like Cytokines

BACKGROUND & AIMS Concanavalin A (con A)-induced hepatitis is an immunomediated disease in which assembly of CD4(+) T cells and T helper (Th)1-like cytokines causes Fas-mediated liver cell death. Nitric oxide (NO) modulates Th1 response in vitro. NCX-4016 is an NO-aspirin derivative that spares the gastrointestinal tract and shares molecular targets with NO. The aim of this study was to investigate whether this NO-aspirin modulates Th1-like response induced by con A. METHODS BALB/c mice were injected with 0.3 mg con A per mouse alone or in combination with NO-aspirin (18-100 mg/kg) or aspirin (10-55 mg/kg). RESULTS NO-aspirin, but not aspirin, caused a dose-dependent protection against liver damage induced by con A. At a dose of 100 mg/kg, NO-aspirin caused a 40%-80% reduction of interleukin (IL)-1beta, IL-12, IL-18, interferon (IFN)-gamma, and tumor necrosis factor alpha production without affecting cytokine messenger RNA expression. NO-aspirin prevented Fas, Fas ligand, and IL-2 receptor up-regulation on spleen lymphocytes and Fas ligand on hepatocytes and caused the S-nitrosylation/inhibition of IL-1beta-converting enzyme-like cysteine proteases (caspases) involved in the processing and maturation of IL-1beta and IL-18. IL-18 immunoneutralization prevented IFN-gamma release and protected from liver injury induced by con A. In contrast to a selective caspase 1 inhibitor, zVAD.FMK, a pancaspase inhibitor, prevented IFN-gamma release and protected the liver from injury. CONCLUSIONS Th1-like response induced by con A is mediated by IL-18 and requires activation of multiple caspases. NCX-4016 causes the S-nitrosylation/inhibition of caspases involved in cytokine production. Inhibition of Th1-like response is a new anti-inflammatory mechanism of action of NO-aspirin.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

: Nitric oxide (NO)‐releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO‐NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown.

TNFalpha processing enzyme inhibitors prevent aspirin-induced TNFalpha release and protect against gastric mucosal injury in rats.

Although previous studies indicate that prevention of tumour necrosis factor α (TNFα) release protects against NSAID‐induced gastric mucosal injury, intracellular pathways by which aspirin causes TNFα release are unknown. TNFα is synthesized as a precursor which is proteolytically cleaved by a specific converting enzyme, TACE, to release the mature cytokine. TACE inhibitors prevent TNFα release and protect against TNFα‐mediated disease.

NSAIDs upregulate β2‐integrin expression on human neutrophils through a calcium‐dependent pathway

Margination of circulating neutrophils (PMN) into the gastric microcirculation is an early and critical event in the pathogenesis of non‐steroidal antinflammatory drug (NSAID)‐induced gastropathy. This effect is mediated through the upregulation of β2 integrins on the PMN surface.

Bombesin-induced pancreatic regeneration in pigs is mediated by p46Shc/p52Shc and p42/p44 mitogen-activated protein kinase upregulation.

BACKGROUND In several animal species the pancreas has the capacity to partially regenerate in a self regulating process. A complex network of growth factors modulates this process. There is evidence that bombesin stimulates pancreatic regeneration in rodents. Whether bombesin stimulates pancreas regrowth in large mammals is unknown. Shc proteins, the target of tyrosine kinase-coupled receptors, activate p42 and p44 mitogen-activated protein (MAP) kinase and induce the transcriptional upregulation of genes involved in cell proliferation. The aims of our study were to determine whether bombesin stimulates pancreatic growth in large mammals and whether this event requires Shc-MAP kinase pathway upregulation. METHODS Three groups of pigs were submitted to sham operation (group 1); to subtotal (70%) distal pancreatectomy (group 2), and to subtotal pancreatectomy followed by bombesin (5 mg three times daily) for 4 weeks (group 3). After a 4-week follow-up a second laparotomy was performed, and the residual pancreas removed. p46Shc, p52Shc and p66Shc, Grb2, and p42/p44 MAP kinase expression and phosphorylation were measured either in freshly isolated pancreatic acinar cells or whole pancreatic extracts. RESULTS In vivo bombesin administration resulted in: 1) approximately 100% growth of pancreatic duodenal lobe; 2) rapid recovery from exocrine pancreatic failure; and 3) a threefold increase in the rate of pancreatic acinar cell proliferation. Incubating freshly isolated pancreatic acinar cells with bombesin resulted in time- and concentration-dependent stimulation of p46Shc/p52Shc phosphorylation, Shc-Grb2 complex formation, and p42/p44 MAP kinase activation. In vivo bombesin administration significantly upregulated p46Shc/p52Shc and MAP kinase expression and/or activity in whole pancreatic extracts. CONCLUSIONS In vivo chronic bombesin administration stimulates pancreatic regeneration after pancreatectomy in large mammals. Bombesin-stimulated pancreatic growth is associated with upregulation of the Shc-Grb2-SOS-Ras-MAP kinase pathway.

Prevention of gastric damage by no-releasing aspirin (NCX-4016) is mediated by inhibition of ICE/-like proteases in gastric mucosa and endothelial cells

Background. Tumor necrosis factor-ct (TNFtx), a proinflammatory cytokine, is released in vivo and in vitro after exposure to non-steroidal anti-inflammatory drugs (NSAIDs). TNFct receptor binding leads to activation of multiple interleukin-1 converting enzyme (ICE)-like proteases (caspases) and endothelial cell death. Endothelial cell dysfunction is an early step in the process of NSAID-gastropathy. Nilric oxide(NO)-releasing NSAIDs, are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. NO-releasing agents (sodium nitroprusside [SNP] and L-Arginine) prevent TNF-induced ICE/caspase 1 activation in human endothelial ceils (HUVECs). Aims. To assess whether NO-NSAIDs modulate ICE-like proteases in vivo and in vitro and protect endothelial cells from TNFct-induced apoptosis. Methods. Macrophages, prepared from mouse spleen, were used to investigate TNFet release in vitro. Gastric damage was induced in rat by oral administration of 150 mg/kg aspirin or an equimolar dose (249 mg/kg) of NCX-4016 (NicOx, Milan, Italy). ICE-like activity was measured in cell Iysates and gastric homogenates using YVAD.AMC as substrate in a fluorimetric assay. HUVEC apoptosis was assessed by FACS-scan analysis. Results. In vitro studies demonstrated that aspirin, but not NCX-4016, released TNFct from mouse macrophages. Co-incubation with 1-100 laM SNP resulted in a concentrationdependent inhibition of aspirin-induced TNFct-release. In viw) aspirin administration caused a time-dependent increase in gastric mucosal damage and gastric mucosa ICE/caspase activity. Pretreating rats with 0.1 mM L-Arginine, SNP and Z.VAD.fmk, a pan-ICE/caspase inhibitor, prevented both gastric mucosal damage and ICE/caspase upregulation induced by aspirin. No gastric damage was detectable with any dose of NCX-4016 tested. Like aspirin NCX-4016 was largely metabolized to salicylate. In contrast to aspirin NCX-416 significantly increased plasma nitrite/nitrate concentrations and inhibited the aspirin-induced upregulation of ICE/caspase activity into the gastric mucosa. Although aspirin had no effect on HUVEC apoptosis induced by TNFcq NCX-4016 and SNP caused a concentrationdependent inhibition of apoptosis and prevented ICEqike pmteases activation as demonstrated by Westem blot analysis and fluorimetric assay. NCX-4016 inhibited IL-113 release from endotoxin-stimulated macrophages. Conclusions. 1) Activation of ICE/caspase pmteases is a key step in the process of NSAID-gastropathy; 2) NCX-4016 spares the gastric mucosa and protects endothelial cells from TNFetinduced apoptosis. The potential of NO-releasing NSAIDs to modulate ICE-like pmteases and IL-113 release might help to explain the anti-inflammatory activity of these compounds.