Local and systemic delivery of a novel group of inhibitors of transglutaminase enzyme: A potential approach for treating of catheter-related complications and liver fibrosis

The present thesis investigates targeted (locally and systemically) delivery of a novel group of inhibitors of enzyme transglutaminases (TGs). TGs are a widely distributed group of enzymes that catalyse the formation of isopeptide bonds between the y-carboxamide group of protein-bound glutamines and the a-amino group of protein-bound lysines or polyamines. The first group of the novel inhibitors tested were the tluorescently labelled inhibitors of Factor XIIIa (FXIIIa). These small, non-toxic inhibitors have the potential to prevent stabilisation of thrombi by FXIIIa and consequently increase the natural rate of thrombolysis, in addition it reduces staphylococcal colonisation of catheters by inhibiting their FXIIIa¬mediated cross-linking to blood clot proteins on the central venous catheter (CVCs) surface. The aim of this work was to incorporate the FXIIIa inhibitor either within coating of polyurethane (PU) catheters or to integrate it into silicone catheters, so as to reduce the incidence of thrombotic occlusion and associated bacterial infection in CVCs. The initial work focused on the incorporation of FXIIIa inhibitors within polymeric coatings of PU catheters. After defining the key characteristics desired for an effective polymeric-coating, polyvinylpyrrolidone (PVP), poly(lactic-co-glycolic acid) (PLGA) or their combination were studies as polymers of choice for coating of the catheters_ The coating was conducted by dip-coating method in a polymer solution containing the inhibitor. Upon incubation of the inhibitor-and polymer-coated strips in buffer, PVP was dissolved instantly, generating fast and significant drug release, whilst PLGA did not dissolve, yielding a slow and an insufficient amount of drug release. Nevertheless, the drug release profile was enhanced upon employing a blend solution of PVP and PLGA. The second part of the study was to incorporate the FXIIIa inhibitor into a silicone elastomer; results demonstrated that FXIIIa inhibitor can be incorporated and released from silicone by using citric acid (CA) and sodium bicarbonate (SB) as additives and the drug release rate can be controlled by the amount of incorporated additives in the silicone matrix. Furthermore, it was deemed that the inhibitor was still biologically active subsequent to being released from the silicone elastomer strips. Morphological analysis confirmed the formation of channels and cracks inside the specimens upon the addition of CA and SB. Nevertheless, the tensile strength, in addition to Young's modulus of silicone elastomer strips, decreased constantly with an increasing amount of amalgamated CA/ SB in the formulations. According to our results, incorporation of FXIIIa inhibitor into catheters and other medical implant devices could offer new perspectives in preventing bio-material associated infections and thrombosis. The use of tissue transglutaminase (T02) inhibitor for treating of liver fibrosis was also investigated. Liver fibrosis is characterized by increased synthesis and decreased degradation of the extracellular matrix (ECM). Transglutaminase-mediated covalent cross-linking is involved in the stabilization of ECM in human liver fibrosis. Thus, TG2 inhibitors may be used to counteract the decreased degradation of the ECM. The potential of a liposome based drug delivery system for site specific delivery of the fluorescent TG2 inhibitor into the liver was investigated; results indicated that the TG2 inhibitor can be successfully integrated into liposomes and delivered to the liver, therefore demonstrating that liposomes can be employed for site-specific delivery of TG2 inhibitors into the liver and TG2 inhibitor incorporating liposomes could offer a new approach in treating liver fibrosis and its end stage disease cirrhosis.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

The Rover Task Force:a case study in proactive and reactive policy intervention?

The paper examines the policy responses in the UK West Midlands to the successive crises at the car maker MG-Rover. Whilst the firm’s eventual collapse in 2005 was a substantial shock to the West Midlands economy, the impact was much less than was anticipated when the firm was first threatened with closure in 2000 at the time of its break-up and sale by the German car firm BMW. Although the firm struggled as an independent producer, the five years of continued production until 2005 and the work of the initial Rover Task Force (RTF1), enabled many suppliers to adjust and diversify away from their hitherto dependence on MG-Rover resulting in as many as 10,000–12,000 jobs being ‘saved’. This first intervention was later followed by a programme to help ex-workers to find new jobs or re-train and assist supply firms to continue trading in the short term. Examination of the effectiveness of these emergency initiatives enables a wider discussion about the nature of industrial policy in the region and the work of the local regional development agency’s cluster-based approach to economic development and business support. Whilst the actions taken were successful in a number of aspects, there were a number of significant ‘failures’ at both national and local level. The MG-Rover case also illustrates a number of critical issues pertaining to regionally based cluster policies and the organization of cluster management groups where the ‘cluster’ in question not only crosses both administrative and ‘sector’ boundaries but is also subject to the imperatives of the global market car market.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

An assessment of the alcohol content of medicines commonly used in paediatric liver and cystic fibrosis patients

Poster session - Paediatric cystic fibrosis and liver patients can be prescribed a number of different pharmaceutical preparations, often in liquid form - It is not uncommon for alcohol to be present in liquid preparations, often as a solvent - Although the quantity of alcohol present can be low, patients taking a number of alcohol-containing preparations may be at risk of toxicity - It has been found that only a few of the preparations used for both paediatric cystic fibrosis and liver patients contain alcohol - The quantity of alcohol present in these preparations is low and should not cause toxicity, even when the products are used in combination

Sarcoidosis, alveolar β-actin and pulmonary fibrosis

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown aetiology. Proteins present within the alveolar space early in sarcoidosis disease may provide an insight into novel mechanisms for the development of fibrotic disease and in particular pulmonary fibrosis.

METHODS: A modified two-dimensional difference gel electrophoresis protocol was applied to the human bronchoalveolar lavage fluid (hBALF) of four patients with non-persistent pulmonary interstitial disease at 4-year follow-up (defined as mild disease) and four patients who developed pulmonary interstitial disease at 4-year follow-up (defined as severe disease). The protein β-actin was identified by LC-MS/MS from a preparative gel and found to be significantly elevated in early lavages from the severe disease group. To look at the potential pro-fibrotic effects of this protein, primary human pulmonary fibroblasts (CCD-19Lu) were treated with recombinant β-actin following which qPCR and ELISA assays were used to measure any effects.

RESULTS: We found that β-actin levels were significantly elevated in early hBALF samples in patients who subsequently developed severe disease when compared to the mild group. Treating primary human pulmonary fibroblasts with recombinant β-actin led to enhanced gene expression of the pro-fibrotic markers alpha smooth muscle actin and collagen 1 as well as the increased secretion of interleukin-13 and metalloproteinases 3 and 9.

CONCLUSION: Free β-actin within the lungs of sarcoidosis patients potentially may contribute to disease pathogenesis particularly in the context of abnormal remodelling and the development of pulmonary fibrosis.

Lack of Association Between Haptoglobin Phenotype and Cystic Fibrosis Outcomes

Haptoglobin (Hp), a heme-Iron chelator, has different isoforms which are associated with variable tendency toward infections: Hp 1-1, Hp 2-1, and Hp 2-2. Cystic fibrosis (CF) outcomes are variable and influenced by genetic and environmental factors. The aim of this study was to determine whether Hp phenotype influenced disease severity in CF. One hundred forty-two CF patients from two centers were analyzed for Haptoglobin phenotype using gel electrophoresis of hemoglobin enriched serum. Clinical and microbiological data including bacterial colonization status, lung function, presence of CF-related diabetes and liver disease, rate of exacerbation, and mortality were compared between Hp phenotype groups. We found a trend toward less mucoid PA among Hp 2-2 (20.4 %) compared with Hp 1-1 and Hp 2-1 individuals (33.3 %), p = 0.317. Hp 2-2 individuals also had less antibiotic courses, and lower inflammatory markers without statistical significance. Haptoglobin phenotype is unlikely to be an important modifier of CF phenotype.

Cirrhosis Diagnosis and Liver Fibrosis Staging: Transient Elastometry Versus Cirrhosis Blood Test.

International audience

New insights into plant salt acclimation: the roles of vesicle trafficking and reactive oxygen species signalling in mitochondria and the endomembrane system

In this study, we investigated the cellular and molecular mechanisms that regulate salt acclimation. The main objective was to obtain new insights into the molecular mechanisms that control salt acclimation. Therefore, we carried out a multidisciplinary study using proteomic, transcriptomic, subcellular and physiological techniques. We obtained a Nicotiana tabacum BY-2 cell line acclimated to be grown at 258 mM NaCl as a model for this study. The proteomic and transcriptomic data indicate that the molecular response to stress (chaperones, defence proteins, etc.) is highly induced in these salt-acclimated cells. The subcellular results show that salt induces sodium compartmentalization in the cell vacuoles and seems to be mediated by vesicle trafficking in tobacco salt-acclimated cells. Our results demonstrate that abscisic acid (ABA) and proline metabolism are crucial in the cellular signalling of salt acclimation, probably regulating reactive oxygen species (ROS) production in the mitochondria. ROS may act as a retrograde signal, regulating the cell response. The network of endoplasmic reticulum and Golgi apparatus is highly altered in salt-acclimated cells. The molecular and subcellular analysis suggests that the unfolded protein response is induced in salt-acclimated cells. Finally, we propose that this mechanism may mediate cell death in salt-acclimated cells.

Structural and cellular characterization of Idiopathic Pulmonary Fibrosis

La Fibrosi Polmonare Idiopatica (IPF) è una malattia polmonare cronica, irreversibile la cui eziologia risulta essere ignota, caratterizzata da un processo fibrotico progressivo che inizia nel tratto respiratorio inferiore. Le persone affette da IPF presentano età media compresa tra 55 e 77 anni. L’incidenza annuale di IPF è stata recentemente stimata tra 14 e 42,7 casi per 100.000 persone e tale dato risulta essere in aumento. IPF fa parte delle malattie Polmonari Idiopatiche Interstiziali (IIP) che comprendono patologie con quadri istologici e clinici differenti. Le affezioni su cui si concentrerà questo studio sono: UIP (Usual Interstitial Pneumonia) caratterizzata da fibrosi interstiziale e dalla presenza di foci fibrotici connessi alla pleura e corrispondente al quadro anatomopatologico della maggior parte dei casi di IPF; NSIP (Non Specific Interstitial Pneumonia) simile alla UIP ma con maggiore uniformità temporale e spaziale delle manifestazioni; Sarcoidosi, malattia granulomatosa ad eziologia ignota. Attualmente la gravità della IPF, che implica una mortalità del 50% dei pazienti a 5 anni dall’esordio, e la scarsa efficacia farmacologica nel rallentarne la progressione vedono il trapianto polmonare come unica possibilità di sopravvivenza nelle forme più severe. Al momento non è chiaro il meccanismo patogenetico di insorgenza e progressione della IPF anche se sono stati individuati alcuni fattori scatenanti quali fumo di sigaretta, infezioni respiratorie e inquinanti atmosferici; tuttavia nessuno di tali elementi può da solo determinare un così esteso e progressivo rimodellamento del parenchima polmonare. Numerose sono le evidenze di come il substrato genetico, le alterazioni del rapporto morte/proliferazione cellulare e le citochine svolgano un ruolo nella genesi e nella progressione della malattia, ma non sono ancora chiari i fenomeni biologico-cellulari che la sostengono e, quindi, quali siano i punti di attacco per poter incidere terapeuticamente nel modificare l’evoluzione della IPF. Poiché il nostro laboratorio ha partecipato alla scoperta dell’esistenza di cellule staminali nel polmone umano normale, uno degli obiettivi finali di questo progetto si basa sull’ipotesi che un’alterazione del compartimento staminale svolga un ruolo cruciale nella eziopatogenesi di IPF. Per questo in precedenti esperienze abbiamo cercato di identificare nella IPF cellule che esprimessero antigeni associati a staminalità quali c-kit, CD34 e CD133. Questo lavoro di tesi si è proposto di condurre un’indagine morfometrica ed immunoistochimica su biopsie polmonari provenienti da 9 pazienti affetti da UIP, 3 da NSIP e 5 da Sarcoidosi al fine di valutare le alterazioni strutturali principali imputabili alle patologie. Preparati istologici di 8 polmoni di controllo sono stati usati come confronto. Come atteso, è stato osservato nelle tre patologie esaminate (UIP, NSIP e Sarcoidosi) un significativo incremento nella sostituzione del parenchima polmonare con tessuto fibrotico ed un ispessimento dei setti alveolari rispetto ai campioni di controllo. L’analisi dei diversi pattern di fibrosi presenti fa emergere come vi sia una netta differenza tra le patologie con una maggiore presenza di fibrosi di tipo riparativo e quindi altamente cellulata nei casi di UIP, e NSIP mentre nelle Sarcoidosi il pattern maggiormente rappresentato è risultato essere quello della fibrosi replacement o sostitutiva. La quantificazione delle strutture vascolari è stata effettuata tenendo separate le aree di polmone alveolare rispetto a quelle occupate da focolai sostitutivi di danno (componente fibrotica). Nei campioni patologici analizzati era presente un significativo riarrangiamento di capillari, arteriole e venule rispetto al polmone di controllo, fenomeno principalmente riscontrato nel parenchima fibrotico. Tali modifiche erano maggiormente presenti nei casi di NSIP da noi analizzati. Inoltre le arteriole subivano una diminuzione di calibro ed un aumento dello spessore in special modo nei polmoni ottenuti da pazienti affetti da Sarcoidosi. Rispetto ai controlli, nella UIP e nella Sarcoidosi i vasi linfatici risultavano inalterati nell’area alveolare mentre aumentavano nelle aree di estesa fibrosi; quadro differente si osservava nella NSIP dove le strutture linfatiche aumentavano in entrambe le componenti strutturali. Mediante indagini immunoistochimiche è stata documentata la presenza e distribuzione dei miofibroblasti, positivi per actina muscolare liscia e vimentina, che rappresentano un importante componente del danno tissutale nella IPF. La quantificazione di questo particolare fenotipo è attualmente in corso. Abbiamo inoltre analizzato tramite immunoistochimica la componente immunitaria presente nei campioni polmonari attraverso la documentazione dei linfociti T totali che esprimono CD3, andando poi a identificare la sottopopolazione di T citotossici esprimenti la glicoproteina CD8. La popolazione linfocitaria CD3pos risultava notevolmente aumentata nelle tre patologie analizzate soprattutto nei casi di UIP e Sarcoidosi sebbene l`analisi della loro distribuzione tra i vari distretti tissutali risultasse differente. Risultati simili si sono ottenuti per l`analisi dei linfociti CD8pos. La componente monocito-macrofagica è stata invece identificata tramite la glicoproteina CD68 che ha messo in evidenza una maggiore presenza di cellule positive nella Sarcoidosi e nella UIP rispetto ai casi di NSIP. I dati preliminari di questo studio indicano che il rimodellamento strutturale emo-linfatico e cellulare infiammatorio nella UIP si differenziano rispetto alle altre malattie interstiziali del polmone, avanzando l’ipotesi che il microambiente vascolare ed immunitario giochino un ruolo importante nella patogenesi della malattia

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial-mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0-3, 6-9 or 10-13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.

Nanopowder management and plasma parameters in nanofabrication of low-dimensional quantum structures in reactive silane-based plasmas

Management of nanopowder and reactive plasma parameters in a low-pressure RF glow discharge in silane is studied. It is shown that the discharge control parameters and reactor volume can be adjusted to ensure lower abundance of nanopowders, which is one of the requirements of the plasma-assisted fabrication of low-dimensional quantum nanostructures. The results are relevant to micro- and nanomanufacturing technologies employing low-pressure glow discharge plasmas of silane-based gas mixtures.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.