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Telephone triage is increasingly used to manage workload in primary care; however, supporting evidence for this approach is scarce. We aimed to assess the effectiveness and cost consequences of general practitioner-(GP)-led and nurse-led telephone triage compared with usual care for patients seeking same-day consultations in primary care.

This study aimed to determine if the lens protein aquaporin 0 (AQP0) is present in the vitreous of pseudophakic eyes of patients presenting with chronic cystoid macular edema (CME).

Cerebral palsy is one of the most devastating consequences of brain injury around the time of birth, and nearly a third of cases are now associated with premature birth. Compared with term babies, preterm babies have an increased incidence of complications that may increase the risk of disability, such as intraventricular hemorrhage, periventricular leukomalacia, sepsis, and necrotizing enterocolitis. The response to injury is highly dependent on brain maturity, and although cellular vulnerability is well documented, there is now evidence that premyelinating axons are also particularly sensitive to ischemic injury. In this review, we will explore recent evidence highlighting a central role for glia in mediating increased risk of disability in premature infants, including excessive activation of microglia and opening of astrocytic gap junction hemichannels in spreading injury after brain ischemia, in part likely involving release of adenosine triphosphate (ATP) and overactivation of purinergic receptors, particularly in white matter. We propose the hypothesis that inflammation-induced opening of connexin hemichannels is a key regulating event that initiates a vicious circle of excessive ATP release, which in turn propagates activation of purinergic receptors on microglia and astrocytes. This suggests that developing effective neuroprotective strategies for preterm infants requires a detailed understanding of glial responses.

Flood exposure is increasing in coastal cities owing to growing populations and assets, the changing climate, and subsidence. Here we provide a quantification of present and future flood losses in the 136 largest coastal cities. Using a new database of urban protection and different assumptions on adaptation, we account for existing and future flood defences. Average global flood losses in 2005 are estimated to be approximately US$6 billion per year, increasing to US$52 billion by 2050 with projected socio-economic change alone. With climate change and subsidence, present protection will need to be upgraded to avoid unacceptable losses of US$1 trillion or more per year. Even if adaptation investments maintain constant flood probability, subsidence and sea-level rise will increase global flood losses to US$60–63 billion per year in 2050. To maintain present flood risk, adaptation will need to reduce flood probabilities below present values. In this case, the magnitude of losses when floods do occur would increase, often by more than 50%, making it critical to also prepare for larger disasters than we experience today. The analysis identifies the cities that seem most vulnerable to these trends, that is, where the largest increase in losses can be expected.

Brain damage resulting from cerebral ischemia remains a significant problem at all stages of life. In adults, ischemic stroke is the third leading cause of death and the leading cause of disability in the developed world. In term newborns, moderate to severe brain damage after hypoxia-ischemia (HI) occurs in 1-3 per 1000 live births. One of the most striking features of HI injury is that after initial recovery of cellular oxidative metabolism, there is a delayed, ‘secondary’ mitochondrial failure that spreads over time from the most severely damaged areas outwards, into previously undamaged regions. This secondary failure is accompanied by transient seizure activity and cytotoxic edema. The specific mechanisms of this spread are poorly understood, but it is at least partly associated with spreading waves of depression that can trigger cell death in neighboring uninjured tissues. The waves are propagated through cell-cell communication via gap junction channels (the so called “bystander effect”). It has recently been proposed that unopposed connexin hemichannels (connexons) also play a significant role by mediating release of paracrine molecules that in turn propagate cell death messages by releasing intracellular mediators such as ATP, NAD(), or glutamate or by abnormally prolonged opening to allow cell edema. There is increasing evidence that connexin hemichannels contribute to injury after many neural insults and that it is possible to significantly reduce the spread of damage after injury by suppressing the induction or activity of the connexin proteins that form hemichannels.

Antisense oligodeoxynucleotides (AsODN) are rapidly degraded by nucleases in biological fluids which compromises their efficacy as therapeutic agents. This study evaluated two Fluorescence Resonance Energy Transfer (FRET) approaches, Acceptor Photobleaching and Sensitized Emission, in terms of their suitability for quantification of oligonucleotide stability in various colloidal carrier systems in vitro. The influence of the formulations pH and viscosity on the validity of the two approaches was determined and showed that the donor fluorescence intensity was highly susceptible to pH fluctuations of the medium. Moreover, the Acceptor Photobleaching approach proved to be unsuitable for the proposed studies due to Brownian motion of molecules in liquid formulations, suggesting that this method can only be used for immobilized specimens. The stability of a 30-mer AsODN incorporated into various in situ gelling systems was evaluated using the Sensitized Emission approach. This approach appeared to offer a simple tool to evaluate the stability of AsODN and showed stable molecules over a period of one week. However, a number of criteria, such as photobleaching due to repeated exposure, pH of the surrounding medium and sample preparation need to be carefully considered when performing quantitative FRET measurements.

Ion-activated in situgelling systems are able to cross-link with the cations present in the tear fluid, forming a gel on the ocular surface and prolonging corneal contact time. Corneal scrape wounding offers an exceptional model to investigate the efficacy of these formulations for connexin43 (Cx43) antisense oligodeoxynucleotide (AsODN) delivery used to improve wound repair. Systems based on gellan gum and carrageenan have previously been found advantageous in terms of their physicochemical properties, in vitroand in vivorelease profiles and precorneal retention. The present study describes AsODN penetration into corneal tissue after wounding and determines the formulations’ delivery efficacy by evaluating wound size, tissue inflammation and connexin levels. No difference was shown between the penetration patterns of the formulations, with most of the AsODN accumulating in the epithelium close to the wound leading edge and the stroma underlying the wound. However, significant differences were seen in the delivery efficacy, with gellan gum and carrageenan based systems resulting in the lowest connexin levels and subsequently in the greatest reduction in wound size, the least stromal edema and hypercellularity. This demonstrates their potential use as delivery vehicles for AsODNs to the ocular surface.

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 μg/min) produced a positive inotropic effect as demonstrated by significant increases in systolic pressure (P< 0.05) and pressure derivative (dp/dtmax) 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 μM following CORM-3, there was no additional increase in systolic pressure or dp/dtmax. However, significant rises in systolic pressure and dp/dtmax 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.

The development of mAbs remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies. Often, the only relevant species for preclinical safety assessment of mAbs are non-human primates (NHPs), and this raises important scientific, ethical and economic issues. To investigate evidence-based opportunities to minimize the use of NHPs, an expert working group with representatives from leading pharmaceutical and biotechnology companies, contract research organizations and institutes from Europe and the USA, has shared and analyzed data on mAbs for a range of therapeutic areas. This information has been applied to hypothetical examples to recommend scientifically appropriate development pathways and study designs for a variety of potential mAbs. The addendum of ICHS6 provides a timely opportunity for the scientific and regulatory community to embrace strategies which minimize primate use and increase efficiency of mAb development.

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 µM) 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.

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.

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 (HbA(0)) 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 HbA(0) 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.

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-PK1cells to CP (50 μM) caused significant apoptosis as evidenced by caspase-3 activation and an increased number of floating cells. Treatment with CORM-3 (1–50 μM) 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.

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.

The induction of heme oxygenase-1 (HO-1) is widely recognized as an effective cellular strategy to counteract a variety of stressful events. We have shown that curcumin and caffeic acid phenethyl ester, two naturally occurring phytochemicals that possess antioxidant, anti-inflammatory, and anticarcinogenic activities, induce HO-1 in many cell types. This suggests that stimulation of HO-1 could partly underlie the beneficial effects exerted by these plant-derived constituents. Here we examined the ability of additional plant constituents to up-regulate heme oxygenase activity and HO-1 in aortic endothelial cells. Incubation of endothelial cells with a series of polyphenolic chalcones (5-50 muM) resulted in increased heme oxygenase activity; interestingly, the chemical structure dictated the pattern of heme oxygenase induction, which was unique to each particular compound employed. We also found that rosolic acid, a constituent isolated from the rhizome of Plantago asiatica L. dramatically increased HO-1 in a concentration- and time-dependent manner. Severe cytotoxicity was observed after prolonged exposure (24 or 48 h) of cells to curcumin and caffeic acid phenethyl ester, whereas 2'-hydroxychalcone and rosolic acid did not affect cell viability. By using different mitogen-activated protein kinase inhibitors, we determined that the extracellular signal-regulated kinase, p38, and c-Jun NH(2)-terminal protein kinase pathways play only a minor role in the induction of HO-1 by rosolic acid and 2'-hydroxychalcone. On the other hand, increased intra- and extracellular thiols markedly reduced the rise in heme oxygenase activity elicited by rosolic acid. Thus, this study identified novel plant constituents that highly induce HO-1 in endothelial cells and investigated some of the mechanisms involved in this effect.

The family of haem oxygenase enzymes is unique in nature for its role in haem degradation. Haem is cleaved at the a-meso position by haem oxygenase with the support of electrons donated by cytochrome P450 reductase, the first products of this reaction being CO, iron and biliverdin. Biliverdin is then converted to bilirubin by biliverdin reductase. If haem is viewed as a substrate for an anabolic pathway, it becomes evident that haem oxygenases do not break down haem for elimination from the body, but rather use haem to generate crucial molecules that can modulate cellular functions. The facts that biliverdin and bilirubin are potent antioxidants and that CO is both a vasoactive and signalling molecule sustain this idea. The existence of a constitutive haem oxygenase (HO-2), mainly present in the vasculature and nervous system, and an inducible haem oxygenase (HO-1), which is highly expressed during stress conditions in all tissues, also suggests that cells have evolved a fine control of this enzymic pathway to ultimately regulate haem consumption and to ensure production of CO, biliverdin/bilirubin and iron during physiological and pathophysiological situations. This review will focus primarily on the biological actions of biliverdin and bilirubin derived from the haem oxygenase/biliverdin reductase systems and their potential roles in counteracting oxidative and nitrosative stress.

Carbon monoxide (CO) is a resourceful gas as recent advances in the area of cell signaling are revealing an unexpected physiological role for CO in the cardiovascular, immune and nervous systems. Transition metal carbonyls have been lately discovered to function as COreleasing molecules (CO-RMs) and elicit distinct pharmacological activities in biological systems. Studies currently ongoing in our laboratories are investigating both the chemical and bioactive features of a series of water-soluble CO-RMs and their specific utilization as vasoactive mediators, anti-inflammatory agents and inhibitors of cellular and tissue damage. The data presented in this review corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO in mammals and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cellular and organ dysfunction.

It is now established that NO is a messenger molecule in mammals despite its high toxicity. As NO⁺ and CO are isoelectronic, it should not be unexpected that CO could also have a role as a messenger. CO is produced naturally in humans at a rate of between 3 and 6 cm³ per day, and this rate is increased markedly by certain inflammatory states and pathological conditions associated with red blood cell hemolysis. Over the last 10 years, the interest in the biological effects of CO has greatly increased, and it is now established in the medical literature that CO does have a major role as a signaling molecule in mammals. It is particularly active within the cardiovascular system, for example, in suppressing organ graft rejection and protecting tissues from ischemic injury and apoptosis. Recently it has been shown that metal carbonyls can also function as CO-releasing molecules and provide similar biological activities. This opens the possibility to develop pharmaceutically important metal carbonyls.

Mittlerweile steht fest, dass NO trotz seiner hohen Toxizität in Säugetieren als Botenstoff fungiert. Da NO⁺ und CO isoelektronisch sind, sollte es nicht überraschen, dass CO eine gleichartige Funktion zukommt. Der Mensch produziert selbst etwa 3–6 cm³ CO pro Tag, wobei bestimmte Entzündungszustände sowie pathologische Bedingungen, die mit der Hämolyse der roten Blutkörperchen verbunden sind, die produzierte Menge merklich erhöhen. In den letzten zehn Jahren ist das Interesse an den biologischen Wirkungen von CO stark gestiegen und inzwischen gilt es in der medizinischen Literatur als gesichert, dass CO in Säugetieren eine wesentliche Rolle als Signalmolekül spielt. Dabei ist der Hauptwirkungsbereich das Herz-Kreislauf-System, z. B. bei der Unterdrückung von Abstoßungsreaktionen bei Organtransplantationen und beim Schutz des Gewebes vor ischämischer Schädigung und Apoptose. Vor kurzem wurde gezeigt, dass Carbonylmetallkomplexe im Organismus CO freisetzen und ähnliche biologische Wirkungen wie CO selbst zeigen können. Dies eröffnet die Möglichkeit, pharmazeutisch wichtige Carbonylmetallkomplexe zu entwickeln.

NO potently up-regulates vascular haem oxygenase-1 (HO-1), an inducible defensive protein that degrades haem to CO, iron and the antioxidant bilirubin. Since several pathological states are characterized by increased NO production and liberation of haem from haem-containing proteins, we examined how NO influences HO-1 induction mediated by haemin. Aortic endothelial cells treated with S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP) or diethylenetriamine-NONOate (DETA/NO) and haemin exhibited higher levels of haem oxygenase activity compared with cells exposed to NO donors or haemin alone. This was accompanied by a marked increase in bilirubin production and, notably, by a strong magnification of cellular haem uptake. A role for haem metabolites in modulating HO-1 expression by NO was assessed by exposing cells to SNAP, SNP or DETA/NO in medium derived from cells treated with haemin, which contained increased bilirubin levels. This treatment considerably potentiated HO-1 expression and haem oxygenase activity mediated by NO and the use of a haem oxygenase inhibitor abolished this effect. Both iron liberated during haem breakdown and the formation of nitroxyl anion from NO appeared to partially contribute to the amplifying phenomenon; in addition, medium from haemin-treated cells significantly augmented the release of NO by NO donors. Thus we have identified novel mechanisms related to the induction of HO-1 by NO indicating that the signalling actions of NO vary significantly in the presence of haem and haem metabolites, ultimately increasing the defensive abilities of the endothelium to counteract oxidative and nitrosative stress.

Visual analog scales or rating scales are commonly used in economic evaluation to elicit preferences in order to estimate quality-adjusted life years. Values obtained from visual analog scales have been used on their own or via a transformation to map them onto one of the choice-based methods for elicting preferences, namely standard gamble or time trade-off. The arguments against using visual analog scales in economic evaluation directly or indirectly via a transformation are reviewed. It is concluded that it canonly ever provide a second best solution compared with the direct use of a choice-based technique.

The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2–Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2–Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway.

Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18α-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 μM 18α-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18α-GCA for only the first 24–48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18α-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18α-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication. J. Cell. Physiol. 193: 233–243, 2002. © 2002 Wiley-Liss, Inc.

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.

Regulation of IL-1 bioactivity by the IL-1 receptor antagonist (IL-1Ra) is critical for the preservation of normal vascular structure and function in mice. In humans, IL-1 bioactivity may be further fine-tuned by genetic polymorphisms. We recently proposed that an intronic polymorphism of the human gene encoding the IL-1R antagonist (IL1RN) is a genetic risk factor for the development of chronic cardiac allograft rejection. Here we aimed to establish a physiological basis for this susceptibility. Plasma and peripheral blood mononuclear cells (PBMC) were obtained from 55 healthy human volunteers, whose genotypes for four polymorphisms of IL1 family genes (IL1B, IL1R1 and IL1RN) were determined by PCR. IL-1β and IL-1Ra production and release in PBMC cultures were studied by flow cytometry and ELISA. Functional and genotypic data were pooled and analyzed first by multivariate and, subsequently, by univariate statistical tests. Wewere able to confirm our observed association of IL1RN genotype with chronic cardiac allograft rejection using multivariate statistics. IL1RN genotype also emerged as the principal regulator of both constitutive and stimulated IL-1Ra and IL-1β release. Allele IL1RN*;2 was consistently associated with higher IL-1Ra and lower IL-1β release, in a dosage-dependent manner. Conversely, IL1RN*;2 carriage was associated with reduced production of the intracellular isoform of IL-1Ra. These genotypic effects were only observed with prolonged culture prior to stimulation and werenot appreciably influenced by the presence of exogenous modulators of IL-1β production. We conclude that IL1RN genotype is the principal determinant of IL-1β bioactivity within theIL1 gene cluster in humans and discuss its putative role in disease.

Heme oxygenase-1 (HO-1) is an inducible stress protein the expression of which can be markedly augmented in eukaryotes by a wide range of substances that cause a transient change in the cellular redox state. The importance of this protein in physiology and disease is underlined by the versatility of HO-1 inducers and the functional role attributed to HO-1 products (carbon monoxide and bilirubin) in conditions that are associated with moderate or severe cellular stress. An intriguing aspect is the recent evidence showing that nitric oxide, a ubiquitous signaling molecule, finely modulates the activation of HO-1 expression. As the effects of oxidative stress on the regulation of the HO-1 gene have been well established and characterized, this review will focus on the biological relevance of redox signals involving nitric oxide and reactive nitrogen species that lead to up-regulation of the HO-1 pathway, with particular emphasis on vascular tissues and the cardiovascular system.

Hypoxia, cytokines, and nitric oxide (NO) stimulate the generation of vascular endothelial growth factor (VEGF) and induce heme oxygenase-1 (HO-1) expression in vascular tissue. HO-1 degrades heme to carbon monoxide (CO), iron, and biliverdin, the latter being reduced to bilirubin by biliverdin reductase. In the present study, we investigated the role of HO-1 in the modulation of VEGF synthesis in rat vascular smooth muscle cells (VSMC). In VSMC stimulated with cytokines, inhibition of NO production significantly, but not completely, reduced VEGF release. In contrast, inhibition of HO activity by tin protoporphyrin IX (SnPPIX) totally prevented cytokine-induced increase in VEGF, despite an augmented synthesis of intracellular NO. Stimulation of HO-1 activity by hemin enhanced VEGF production; this effect was abrogated by blockade of the HO pathway. Similarly, VEGF synthesis induced by hypoxia was down-regulated by SnPPIX, but not by inhibitors of NO synthase. To elucidate further a direct involvement of HO-1 in the observed effects, we generated transfected cells that overexpressed the HO-1 gene. Notably, these cells synthesized significantly more VEGF protein than cells transfected with a control gene. Among the products of HO-1, biliverdin and bilirubin showed no effect, whereas iron ions inhibited VEGF synthesis. Exposure of cells to 1% CO resulted in a marked accumulation of VEGF (20-fold increase) over the basal level. Our data indicate that HO-1 activity influences the generation of VEGF in VSMC in both normoxic and hypoxic conditions. As CO and iron, respectively the inducer and the inhibitor of VEGF synthesis, are concomitantly produced during the degradation of heme, these data indicate that HO by-products may differentially modulate VEGF production.

Chronic rejection is a leading cause of graft loss in thoracic transplant recipients. Studies on the pathogenesis of chronic rejection have suggested a contributory role for certain cytokines and growth factors. The activity of these mediators is subject to genetic variation if a polymorphism alters expression, or function, of the ligand or its receptor. Here we have asked if certain cytokine and growth factor gene polymorphisms correlate with chronic rejection in recipients of thoracic allografts. In a retrospective analysis of 179 recipients of thoracic organ transplants (128 heart; 36 heart-lung; and 15 lung), polymorphisms in 8 genes that influence the inflammatory process, namely IL1B, IL1R1, IL1RN, IL6, IL10, TNFA, TGFB1and FCGRIIA, were examined. Genotypic data from recipients who had either died or been re-transplanted as a result of chronic rejection (n = 96) were then compared to those of recipients who had a functioning graft for more than 11 years (n = 83). In the heart graft recipients, only those polymorphisms that influenced expression of the IL1receptor antagonist gene had a significant correlation with graft survival, with homozygosity for the IL1RN*1allele being associated with rejection. The alternative, less frequent IL1RNalleles emerged as genomic predictors of long-term allograft survival. This association was especially strong when IL1region haplotypes were considered, particularly when analysis was confined to heart transplant recipients who had had multiple acute rejection episodes (OR > 20). This case-control study indicates that gene polymorphisms which influence IL1 bioactivity also influence the progression of chronic rejection in heart grafts.

The disrupted metabolism of homocysteine (Hcy) causes hyperhomocysteinemia, a condition associated with the impairment of nitric oxide (NO) bio‐availability, tissue hypoxia and increased risk of vascular disease. Here, we examined how Hcy modulates the induction of the stress protein haem oxygenase‐1 (HO‐1) evoked by NO releasing agents and hypoxia in vascular endothelial cells. We found that Hcy (0.5 mM) markedly reduced the increase in haem oxygenase activity and HO‐1 protein expression induced by sodium nitroprusside (SNP, 0.5 mM) but did not affect HO‐1 activation mediated by S‐nitroso‐N‐acetyl‐penicillamine. Cells pre‐treated with Hcy followed by addition of fresh medium containing SNP still exhibited an augmented haem oxygenase activity. Interestingly, high levels of Hcy were also able to abolish hypoxia‐mediated HO‐1 expression in a concentration‐dependent manner. These novel findings indicate that hyperhomocysteinemia interferes with crucial signaling pathways required by cells to respond and adapt to stressful conditions.

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face. © 2001 Wiley-Liss, Inc.

The person trade‐off (PTO) technique has been suggested as a means of eliciting social preferences for health care, both the valuation of health care interventions and, more recently, to inform on the weights that society may attach to other decision‐making criteria (e.g. the severity of a patients pre‐treatment condition). Given the increased attention afforded to the PTO technique, this review examines the current evidence to inform on the ability of the PTO to provide a measure of social preference. Applying criteria of practicality, reliability and validity to empirical and theoretical contributions to the PTO literature, the review finds that the technique has limited empirical support. Applications of the PTO have been in a largely experimental setting, the reliability of the PTO is unproven and the empirical validity of the technique, in terms its ability to reflect actual preferences, remains unclear. In the broader context of the valuation of health states or outcomes, all techniques are open to criticism. Given this position, the review finds support for the potential of the PTO in the assessment of the societal value of health care, and it supports further empirical inquiry on the PTO. Copyright © 2001 John Wiley & Sons, Ltd.

Whilst there is an increased understanding of the cell biology of nerve regeneration, it remains unclear whether there is a direct interrelationship between vascularisation and efficacy of nerve regeneration within a nerve conduit. To establish this is important as in clinical surgery peripheral nerve conduit grafting has been widely investigated as a possible alternative to the use of nerve autografts. The aim of this study was to assess whether vascular endothelial growth factor (VEGF), a highly specific endothelial cell mitogen, can enhance vascularisation and, indirectly, axonal regeneration within a silicone nerve regeneration chamber. Chambers containing VEGF (500–700 ng/ml) in a laminin‐based gel (Matrigel) were inserted into 1 cm rat sciatic nerve defects and nerve regeneration examined in relation to angiogenesis between 5 and 180 d. Longitudinal sections were stained with antibodies against endothelial cells (RECA‐1), axons (neurofilament) and Schwann cells (S‐100) to follow the progression of vascular and neural elements. Computerised image analysis demonstrated that the addition of VEGF significantly increased blood vessel penetration within the chamber from d 5, and by d 10 this correlated with an increase of axonal regeneration and Schwann cell migration. The pattern of increased nerve regeneration due to VEGF administration was maintained up to 180 d, when myelinated axon counts were increased by 78% compared with plain Matrigel control. Furthermore the dose‐response of blood vessel regeneration to VEGF was clearly reflected in the increase of axonal regrowth and Schwann cell proliferation, indicating the close relationship between regenerating nerves and blood vessels within the chamber. Target organ reinnervation was enhanced by VEGF at 180 d as measured through the recovery of gastrocnemius muscle weights and footpad axonal terminal density, the latter showing a significant increase over controls (P< 0.05). The results demonstrate an overall relationship between increased vascularisation and enhanced nerve regeneration within an acellular conduit, and highlight the interdependence of the 2 processes.

The inducible isoform of haem oxygenase (HO-1) has been proposed as an effective system to counteract oxidant-induced cell injury. In several circumstances, this cytoprotective effect has been attributed to increased generation of the antioxidant bilirubin during haem degradation by HO-1. However, a direct implication for HO-1-derived bilirubin in protection against oxidative stress remains to be established. In the present study, we examined the dynamics of HO-1 expression and bilirubin production after stimulation of vascular smooth-muscle cells with hemin, a potent inducer of the HO-1 gene. We found that hemin-mediated increase in HO-1 protein expression and haem oxygenase activity is associated with augmented bilirubin levels. The majority of bilirubin production occurred early after exposure of cells to hemin. Hemin pre-treatment also resulted in high resistance to cell injury caused by an oxidant-generating system. Interestingly, this protective effect was manifest only when cells were actively producing bilirubin as a consequence of increased haem availability and utilization by HO-1. Tin protoporphyrin IX, an inhibitor of haem oxygenase activity, significantly reduced bilirubin generation and reversed cellular protection afforded by hemin treatment. Furthermore, addition of bilirubin to the culture medium markedly reduced the cytotoxicity produced by oxidants. Our findings provide direct evidence that bilirubin generated after up-regulation of the HO-1 pathway is cytoprotective against oxidative stress.