Isothiocyanate-cysteine conjugates protect renal tissue against cisplatin-induced apoptosis via induction of heme oxygenase-1.

Heme oxygenase-1 (HO-1) is a redox sensitive inducible enzyme endowed with important antioxidant and cytoprotective activities. Here we report that two water-soluble isothiocyanate-cysteine conjugates, S-[N-benzyl(thiocarbamoyl)]-l-cysteine (BTTC) and S-[N-(3-phenylpropyl)(thiocarbamoyl)]-l-cysteine (PTTC), potently increase HO-1 protein expression and heme oxygenase activity in renal tubular epithelial cells at 5 and 10µM, while higher concentrations are themselves cytotoxic and pro-apoptotic. Inhibitors of the pro-survival pathways ERK, MAPK and PI3K almost completely abolished the increase in HO-1 induction and heme oxygenase activity, while the JNK pathway appeared to be mainly involved in the apoptosis triggered by the isothiocyanates. We also found that renal cells exposed to 50µM cisplatin (CDDP), a chemotherapeutic agent known for its nephrotoxic actions, displayed a marked increase in caspase-3 activity and the number of apoptotic cells. These effects were abolished by pre-incubation of cells with concentrations of BTCC or PTCC that maximize HO-1 induction and were reversed by the inhibitor of heme oxygenase activity tin protoporphyrin IX (SnPPIX). Moreover, in a model of CDDP-induced nephrotoxicity in vivo, pre-treatment of rats with a daily dose of BTCC or PTCC (25mg/kg, i.p.) completely abolished the increase in serum creatinine and urea levels and markedly reduced the severity of renal tissue apoptosis caused by CDDP. The renoprotective effects of BTCC and PTCC in vivo were markedly attenuated by administration of rats with SnPPIX. These findings indicate that water-soluble isothiocyanates counteract renal dysfunction and apoptosis by up-regulating the HO-1 system and could be used as a supplementary treatment to mitigate CDDP-induced nephrotoxic effects.

Vasorelaxing effects and inhibition of nitric oxide in macrophages by new iron-containing carbon monoxide-releasing molecules (CO-RMs).

Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.

CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke.

OBJECTIVE: Intracerebral hemorrhage is accompanied by a pronounced inflammatory response that mediates brain damage but is also essential for the tissue reparative process. We assessed the effect of CORM-3, a water-soluble carbon monoxide-releasing molecule possessing anti-inflammatory properties, on inflammation and brain injury after intracerebral hemorrhage. DESIGN: In vivo and in vitro laboratory study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats, 250-350 g. INTERVENTIONS: A model of collagenase injection (2 µL) in the brain was established to induce intracerebral hemorrhage. CORM-3 (4 or 8 mg/kg) was administered intravenously at different times as follows: 1) 5 mins before collagenase; 2) 3 hrs after collagenase; and 3) 3 days after collagenase challenge. MEASUREMENTS AND MAIN RESULTS: Saline was used as a negative control. Brain damage, brain water content, and behavioral assessment were evaluated. The inflammatory response was determined at set intervals after intracerebral hemorrhage by counting peripheral neutrophils and lymphocytes, neutrophils, and activated microglia/macrophages in the intracerebral hemorrhage area and measuring plasma tumor necrosis factor-á levels. BV2 microglia and DI-TNC1 astrocytes were exposed to triton (1%) or CORM-3 (10-100 ìM) and cytotoxicity (lactic dehydrogenase assay) measured at 24 hrs. A challenge with collagenase to induce intracerebral hemorrhage caused marked brain damage and modified the levels of inflammatory markers. Pretreatment with CORM-3 significantly prevented injury, modulated inflammation, and reduced plasma tumor necrosis factor-α. CORM-3 given 3 hrs after collagenase significantly increased brain injury and tumor necrosis factor-α production. In contrast, CORM-3 given 3 days after collagenase afforded partial protection, modulated inflammation, and decreased tumor necrosis factor-α starting from the day of application. No dose-dependent effects were observed. CONCLUSIONS: CORM-3 promotes neuroprotection or neurotoxicity after intracerebral hemorrhage depending on the time of administration. Beneficial effects are achieved when CORM-3 is given either before or 3 days after intracerebral hemorrhage, namely, as a prophylactic agent or during the postacute inflammatory phase.

Relationship between leukocyte kinetics and behavioral tests changes in the inflammatory process of hemorrhagic stroke recovery.

In this study, we investigated the inflammatory response to hemorrhagic stroke (HS) as the main mechanism of brain functional recovery. Sprague-Dawley rats (n = 24) underwent surgery with sterile saline (control group, n = 12) and collagenase IV-S (stroke group, n = 12) being injected into the right striatum. White blood cell analysis, histological and immunohistological examination of the brain slices, as well as densitometric analysis of polymorphonuclear and microglial cells/macrophages were correlated with behavioral tests, and the data were subjected to appropriate statistical processing. The results indicate a strong correlation between polymorphonuclear and mononuclear changes in the blood and the zone of hemorrhagic stroke with behavioral tests of functional brain recovery. We propose that the inflammatory response is determined by kinetics of polymorphonuclear and mononuclear cells in both the blood and the hemorrhagic stroke zone. Kinetics of these cells is followed by the restoration of functions, and the maximum functional recovery is observed by the time polymorphonuclear and mononuclear stages have completed. With the development of inflammation and leukocyte kinetics, it is possible to predict functional recovery of hemorrhagic stroke. Improvement of the degree and rate of hemorrhagic stroke functional recovery may be achieved by therapeutic interventions into the inflammatory mechanisms influencing polymorphonuclear and mononuclear cell kinetics.

Human sickle cell blood modulates endothelial heme oxygenase activity: effects on vascular adhesion and reactivity.

OBJECTIVE: Sickle cell disease (SCD) is characterized by extensive hemolysis, increased cellular adhesion, and vaso-occlusion. Tissues from sickle patients express heme oxygenase-1 (HO-1), the enzyme that degrades free heme/hemoglobin to the signaling molecule carbon monoxide, and the antioxidants biliverdin/bilirubin. Here, we examined the HO response in endothelial cells exposed to human sickle blood and determined whether this response is beneficial for SCD. METHODS AND RESULTS: We measured HO activity in human and bovine aortic endothelial cells incubated with human sickle or normal blood. Sickle blood increased HO activity, which was enhanced by hypoxia and was caused mainly by the red cell components of sickle blood. Oxidized hemoglobin was higher in sickle blood and increased markedly over time. Interestingly, HO activity correlated inversely with patients' hemoglobin levels and positively with bilirubin and lactate dehydrogenase. HO-1 induction, exogenous biliverdin, or carbon monoxide markedly decreased adhesion of sickle blood to the endothelium, and sickle red cells partially inhibited relaxation mediated by carbon monoxide in isolated aortas. CONCLUSIONS: Our results highlight important associations between SCD and HO byproducts, which may counteract vascular complications of SCD.

CO liberated from a carbon monoxide-releasing molecule exerts a positive inotropic effect in doxorubicin-induced cardiomyopathy.

Carbon monoxide (CO) liberated by a water-soluble carbon monoxide-releasing molecule (CORM-3) induces a positive inotropic effect with a negative chronotropic effect in normal rat hearts. However, the efficacy of CORM-3 under conditions of chronic cardiac insufficiency is unknown. In a rat model of doxorubicin-induced cardiomyopathy, CORM-3 (20 microg/min) produced a positive inotropic effect as demonstrated by significant increases in systolic pressure (P < 0.05) and pressure derivative (dp/dt max) over time (P < 0.05). A similar dose of CO-depleted negative control (inactive CORM-3) failed to cause any change in these parameters. When the inotrope dobutamine was added at a dose of 10 microM following CORM-3, there was no additional increase in systolic pressure or dp/dt max. However, significant rises in systolic pressure and dp/dt max were observed after dobutamine administration to the hearts previously treated with inactive CORM-3. These results suggest that CORM-3 produces a positive inotropic effect in doxorubicin cardiomyopathy rat hearts, similar to that reported previously in normal hearts. The inotropic effect produced by CO in the doxorubicin cardiomyopathy heart was mimicked by a classical inotrope (dobutamine), suggesting that either a maximal inotropic effect is achieved at this dose of CORM-3 or both drugs utilize shared signaling pathways in cardiac muscle.

Structure-activity relationships of methoxychalcones as inducers of heme oxygenase-1.

Chalcones are naturally occurring flavonoids composed of two aromatic rings connected by a three-carbon unit forming an alpha-beta unsaturated carbonyl group. They are pharmacologically relevant because of their ability to exert anticarcinogenic, antimicrobial, and anti-inflammatory activities. Recent evidence indicates that the bioactivity of hydroxy-chalcones is correlated with their intrinsic property to induce the antioxidant and cytoprotective enzyme heme oxygenase-1 (HO-1). In the present study, we assessed how the methoxy substituents positioned on the two aromatic rings affect the anti-inflammatory action of different chalcones in relation to their ability to increase heme oxygenase in RAW246.7 macrophages. Structure-activity relationships of methoxychlacones were qualitatively and quantitatively examined and correlated with inhibition of endotoxin-mediated nitrite production and cytotoxic effects. Our data indicate that (i) a progressive increase in heme oxygenase activity is obtained by sequentially increasing the number of methoxy substituents in the 3,4,5- and 3',4',5'-positions of the aromatic rings; (ii) methoxy substituents placed either in the 2,4,6-positions or alone in the 4- or 4'-position are ineffective; (iii) increased heme oxygenase activity by chalcones is lost when the alpha-beta double bond and the carbonyl group are reduced or protected; (iv) the anti-inflammatory activity and cytotoxicity profiles of the chalcones examined correlate with their potency as HO-1 inducers; and (v) chalcone-mediated HO-1 induction is reduced by thiols or inhibitors of phosphatidylinositol-3 kinase (PI3K) pathway. This study provides additional information on the structural features that methoxychalcones and natural antioxidants need to possess to be considered as therapeutic agents for maximizing HO-1 expression and activity.

A cytoprotective role for the heme oxygenase-1/CO pathway during neural differentiation of human mesenchymal stem cells.

The inducible protein heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to carbon monoxide (CO) and biliverdin, which play a concerted action in cytoprotection against oxidative stress and in the modulation of cell proliferation and differentiation. Here we report that both HO-1 expression and activity can be highly increased in undifferentiated human mesenchymal stem cells (MSCs) treated with hemin, a known HO-1 inducer. However, HO-1 mRNA and protein expression gradually decrease when MSCs undergo neural differentiation in vitro, making them extremely susceptible to glutamate-mediated cytotoxicity. A time course for HO-1 revealed that this protein is markedly down-regulated after 2 days and returns to control levels 6 days after differentiation. Treatment with glutamate (250 microM) after 2 days of neural differentiation resulted in a more pronounced lactate dehydrogenase release, a marker of cell injury, compared with undifferentiated cells. Notably, cells pretreated with hemin (50 microM) or compounds that release small amounts of CO (10 microM CORM-3 and CORM-A1) rendered cells more resistant to glutamate-induced toxicity; this effect was evident in both undifferentiated and differentiated MSCs. Our findings indicate that MSCs become more vulnerable to oxidative injury during the early stages of differentiation via mechanisms that involve a temporary inhibition of HO-1 expression. Thus, overexpression of HO-1 and CO-releasing molecules could provide a possible therapeutic strategy to improve cell viability during neural differentiation in applications that use stem cell technology.

Improved myocardial function after cold storage with preservation solution supplemented with a carbon monoxide-releasing molecule (CORM-3).

BACKGROUND: Carbon monoxide-releasing molecules (CO-RMs) are pharmacologically active as they protect against cardiac graft rejection and cold ischemia-mediated renal dysfunction. We investigated the cardioprotective role of carbon monoxide (CO) released from CORM-3 against cold ischemia-mediated injury in the heart and evaluated its potential application in the clinical setting of cardiac transplantation. METHODS: Isolated rat hearts underwent cold ischemic storage for 4 or 6 hours using St Thomas Hospital solution that was supplemented with either CORM-3 (50 mumol/liter) or its inactive counterpart (iCORM-3), which does not release CO. Hearts were then reperfused. Both functional parameters and release of cardiac enzymes were assessed. RESULTS: Addition of CORM-3 to the preservation solution resulted in a significant improvement in systolic and diastolic function as well as coronary flow when compared with hearts treated with iCORM-3. In addition, lower levels of the cardiac enzymes creatine kinase and lactate dehydrogenase were measured in the perfusate of hearts stored with CORM-3. CONCLUSIONS: The improved functional recovery and reduced enzyme release after cardiac cold storage with CORM-3, but not iCORM-3, indicate that CO is the main mediator of myocardial protection. Thus, CO-RMs can be used as adjuvants to improve the preservation of hearts for transplantation.

Metal carbonyls as pharmaceuticals? [Ru(CO)3Cl(glycinate)], a CO-releasing molecule with an extensive aqueous solution chemistry.

The pharmacologically active [Ru(CO)(3)Cl(glycinate)] is shown to be in equilibrium with [Ru(CO)(2)(CO(2)H)Cl(glycinate)](-) (isomers) at around pH 3.1 which then at physiological pH reacts with more base to give [Ru(CO)(2)(CO(2))Cl(glycinate)](2-) (isomers) or [Ru(CO)(2)(CO(2)H)(OH)(glycinate)](-) (isomers). The ease with which [Ru(CO)(3)Cl(glycinate)] reacts with hydroxide results in it producing a solution in water with a pH of around 2 to 2.5 depending on concentration and making its solutions more acidic than those of acetic acid at comparable concentrations. Acidification of [Ru(CO)(3)Cl(glycinate)] with HCl gives [Ru(CO)(3)Cl(2)(NH(2)CH(2)CO(2)H)]. The crystal structures of [Ru(CO)(3)Cl(glycinate)] and [Ru(CO)(3)Cl(2)(NH(2)CH(2)CO(2)Me)] are reported.

Curcumin reduces cold storage-induced damage in human cardiac myoblasts.

Curcumin is a polyphenolic compound possessing interesting anti-inflammatory and antioxidant properties and has the ability to induce the defensive protein heme oxygenase-1 (HO-1). The objective of this study was to investigate whether curcumin protects against cold storage-mediated damage of human adult atrial myoblast cells (Girardi cells) and to assess the potential involvement of HO-1 in this process. Girardi cells were exposed to either normothermic or hypothermic conditions in Celsior preservation solution in the presence or absence of curcumin. HO-1 protein expression and heme oxygenase activity as well as cellular damage were assessed after cold storage or cold storage followed by re-warming. In additional experiments, an inhibitor of heme oxygenase activity (tin protoporphyrin IX, 10 microM) or siRNA for HO-1 were used to investigate the participation of HO-1 as a mediator of curcumin-induced effects. Treatment with curcumin produced a marked induction of cardiac HO-1 in normothermic condition but cells were less responsive to the polyphenolic compound at low temperature. Cold storage-induced damage was markedly reduced in the presence of curcumin and HO-1 contributed to some extent to this effect. Thus, curcumin added to Celsior preservation solution effectively prevents the damage caused by cold-storage; this effect involves the protective enzyme HO-1 but also other not yet identified mechanisms.

Evidence for functional inter-relationships between FOXP3, leukaemia inhibitory factor, and axotrophin/MARCH-7 in transplantation tolerance.

In an ex vivo mouse model, regulatory transplantation tolerance is not only linked to Foxp3, but also to release of leukaemia inhibitory factor (LIF) and to expression of axotrophin (also known as MARCH-7), a putative ubiquitin E3 ligase associated with feedback control of T cell activation and of T cell-derived LIF. Given this coordinate correlation with tolerance, we now ask if Foxp3 expression is influenced by LIF or by axotrophin. In spleen cells from allo-rejected mice we found that exogenous LIF reduced interferon gamma release in response to donor antigen by 50%, but LIF had no direct effect on levels of Foxp3 protein in allo-primed cells that were either tolerant, or aggressive, for donor antigen. However, we did find an effect of axotrophin on Foxp3: in the axotrophin null mouse, thymic Foxp3 transcripts were reduced compared to axotrophin wildtype littermates. To test whether these findings in the mouse were of potential significance in man we measured transcript levels of axotrophin and LIF in peripheral blood cell samples collected for a recently published clinical study concerning haematopoietic stem cell recipients. In controls, human peripheral blood CD4+CD25+cells contained significantly more FOXP3 and axotrophin than CD4+CD25-cells. In bone marrow autograft recipients, where peripheral blood cell samples directly represent both the grafted tissue and the immune response, both FOXP3 and axotrophin negatively correlated with graft versus host disease (GVHD). These data suggest that (i) thymic Foxp3+T cell development is influenced by axotrophin; and (ii) clinical auto-GVHD inversely correlates with axotrophin transcript expression as has been previously reported for FOXP3.

Modulation of thrombin-induced neuroinflammation in BV-2 microglia by carbon monoxide-releasing molecule 3.

Carbon monoxide-releasing molecules are emerging as a new class of pharmacological agents that regulate important cellular function by liberating CO in biological systems. Here, we examined the role of carbon monoxide-releasing molecule 3 (CORM-3) in modulating neuroinflammatory responses in BV-2 microglial cells, considering its practical application as a novel therapeutic alternative in the treatment of stroke. BV-2 microglia cells were incubated for 24 h in normoxic conditions with thrombin alone or in combination with interferon-gamma to simulate the inflammatory response. Cells were also subjected to 12 h of hypoxia and reoxygenated for 24 h in the presence of thrombin and interferon-gamma. In both set of experiments, the anti-inflammatory action of CORM-3 was evaluated by assessing its effect on nitric oxide production (nitrite levels) and tumor necrosis factor (TNF)-alpha release. CORM-3 (75 microM) did not show any cytotoxicity and markedly attenuated the inflammatory response to thrombin and interferon-gamma in normoxia and to a lesser extent in hypoxia as evidenced by a reduction in nitrite levels and TNF-alpha production. Inactive CORM-3, which does not liberate CO and is used as a negative control, failed to prevent the increase in inflammatory mediators. Blockade of endogenous CO production by tin protoporphyrin-IX did not change the anti-inflammatory activity of CORM-3, suggesting that CO liberated from the compound is responsible for the observed effects. In addition, inhibition of the mitogen-activated protein kinases phosphatidyl inositol 3 kinase and extracellular signal-regulated kinase amplified the anti-inflammatory effect of CORM-3. These results suggest that the anti-inflammatory activity of CORM-3 could be exploited to mitigate microglia activity in stroke and other neuroinflammatory diseases.

The interaction of nitric oxide with distinct hemoglobins differentially amplifies endothelial heme uptake and heme oxygenase-1 expression.

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.

Protection against cisplatin-induced nephrotoxicity by a carbon monoxide-releasing molecule.

Nephrotoxicity is one of the main side effects caused by cisplatin (CP), a widely used antineoplastic agent. Here, we examined the effect of a novel water-soluble carbon monoxide-releasing molecule (CORM-3) on CP-mediated cytotoxicity in renal epithelial cells and explored the potential therapeutic benefits of carbon monoxide in CP-induced nephrotoxicity in vivo. Exposure of LLC-PK(1) cells to CP (50 microM) caused significant apoptosis as evidenced by caspase-3 activation and an increased number of floating cells. Treatment with CORM-3 (1-50 microM) resulted in a remarkable and concentration-dependent decrease in CP-induced caspase-3 activity and cell detachment. This effect involved activation of the cGMP pathway as 1H-oxadiazole [4, 3-a] quinoxaline-1-ore (ODQ), a guanylate cyclase inhibitor, completely abolished the protection elicited by CORM-3. Using a rat model of CP-induced renal failure, we found that treatment with CP (7.5 mg/kg) caused a significant elevation in plasma urea (6.6-fold) and creatinine (3.1-fold) levels, which was accompanied by severe morphological changes and marked apoptosis in tubules at the corticomedullary junction. A daily administration of CORM-3 (10 mg/kg ip), starting 1 day before CP treatment and continuing for 3 days thereafter, resulted in amelioration of renal function as shown by reduction of urea and creatinine levels to basal values, a decreased number of apoptotic tubular cells, and an improved histological profile. A negative control (iCORM-3) that is incapable of liberating CO failed to prevent renal dysfunction mediated by CP, indicating that CO is directly involved in renoprotection. Our data demonstrate that CORM-3 can be used as an effective therapeutic adjuvant in the treatment of CP-induced nephrotoxicity.

Heme oxygenase-1 mediates the anti-inflammatory actions of 2'-hydroxychalcone in RAW 264.7 murine macrophages.

Chalcones are a group of plant-derived polyphenolic compounds that belong to the flavonoids family, and possess a wide variety of cytoprotective and modulatory functions. Chalcones exert their cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways, such as phase II enzymes and the stress protein heme oxygenase-1 (HO-1). In this study, we investigated the anti-inflammatory action of 2'-hydroxychalcone (2-HC) in a model of lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and examined the role of HO-1 in this process. Our results demonstrate that 2-HC potently induces HO-1 expression and markedly reduces LPS-mediated nitrite and TNF-alpha production. These effects are accompanied by inhibition of inducible nitric oxide synthase protein expression and abolished by blockade of heme oxygenase activity with either tin protoporphyrin IX or HO-1 small interfering RNA. By using a pharmacological approach and siRNA technology, we also found that phosphatidylinositol 3-kinase is a major cellular mediator in 2-HC-induced HO-1 expression. These findings strongly suggest that 2-HC exerts anti-inflammatory actions via activation of the HO-1 pathway and help to elucidate the mechanisms underlying the potential therapeutic value of chalcones.

A carbon monoxide-releasing molecule (CORM-3) attenuates lipopolysaccharide- and interferon-gamma-induced inflammation in microglia.

The development of carbon monoxide-releasing molecules (CO-RMs) in recent years helped to shed more light on the diverse range of anti-inflammatory and cytoprotective activities of CO gas. In this study, we examined the effect of a ruthenium-based water-soluble CO carrier (CORM-3) on lipopolysaccharide (LPS)- and interferon-gamma (INF-gamma)-induced inflammatory responses in BV-2 microglial cells and explored the possible mechanisms of action. BV-2 microglial cells were stimulated with either LPS or INF-gamma in the presence of CORM-3 and the inflammatory response evaluated by assessing the effect on nitric oxide production (nitrite levels) and tumor necrosis factor-alpha (TNF-alpha) release. Similar experiments were also performed in the presence of inhibitors of guanylate cyclase (ODQ), NO synthase (L-NAME), heme oxygenase activity (tin protoporphyrin IX) or various mitogen-activated protein kinase (MAPK) inhibitors. CORM-3 significantly attenuated the inflammatory response to LPS and INF-gamma as evidenced by a significant reduction (p < 0.001) in nitrite levels and TNF-alpha production (P < 0.05). Such effect was maintained in the presence of ODQ, L-NAME or tin protoporphyrin without showing any cytotoxicity. The use of an inactive form of CORM-3 that does not contain carbonyl groups (Ru(DMSO)(4)Cl(2) failed to inhibit the increase in inflammatory markers suggesting that liberated CO mediates the observed effects. In addition, inhibition of phosphatidylinositol-3-phosphate kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways seemed to amplify the anti-inflammatory effect of CORM-3, particularly in cells stimulated with INF-gamma. These results suggest that the anti-inflammatory action of CORM-3 could be exploited to mitigate microglia activation in neuro-inflammatory diseases.

Training in microsurgical skills: Does course-based learning deliver?

Practical skill training courses are an increasingly popular method of teaching surgical skills. Few data are available from instructional courses indicating how successful they are at imparting practical skills to those individuals who attend them. We aimed to identify the skill benefits gained by trainee surgeons attending a 5-day microsurgical skills course. A global scoring system was devised to objectively assess the level of skill employed by trainees to complete an arterial microvascular anastamosis. Vessel patency, anastamotic construct, and care of tissue in the surrounding operative field were taken into account. Postoperative tissue viability and physiological vessel function following anastamosis were also investigated. The majority of surgeons (60%) exhibited an increase in their level of microsurgical skill during the course. The remaining candidates remained static or deteriorated. Attendance at this microsurgical training workshop resulted in an improvement in microsurgical skills in most trainees. In-course assessment by training courses would allow identification of individuals requiring further training or skill refinement.

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages.

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10-100 microM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-alpha, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.

Leukemia inhibitory factor is linked to regulatory transplantation tolerance.

BACKGROUND: The specific regulation of allo-tolerance in vivo occurs within a complex microenvironment and involves co-operation between a small proportion of different cell types within the spleen or draining lymph node. By analyzing unmanipulated whole spleen cell populations we have aimed to mimic this in vivo situation to identify critical signaling molecules in regulatory allo-tolerance. METHODS: We compared the kinetics of cytokine release and induction of signaling proteins in (BALB/c-tolerant)CBA, versus (BALB/c-rejected)CBA, spleen cells after challenge with BALB/c antigen. RESULTS: The distinguishing features of allo-tolerance were Foxp3 protein expression, LIF release, and increased levels of STAT3. Comparison of isogenic clones of Tr1, Th1, and Th2 cells revealed that only the regulatory Tr1 cells are characterized by both LIF and IL10 release. CONCLUSIONS: Overall, our findings demonstrate that allo-antigen driven signaling events can be detected within a whole spleen cell population and identify a role for LIF in the regulation of transplantation tolerance in vivo.