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At this juncture, preclinical and clinical evidence support the adoption of a more restrictive transfusion strategy in most critically ill patients. However, there remain divergent views regarding the risks and benefits of treating anaemia in patients with cardiovascular disease. Laboratory-based studies [68, 69] suggest that patients with cardiovascular disease may require higher haemoglobin concentrations to maintain oxygen delivery in partially occluded or diseased coronary arteries. Studies to demonstrate how anaemia affects contractile function of the left ventricle have rarely shown important effects above haemoglobin concentrations of 70 g/l. Indeed, it is more important to address the underlying pathophysiological causes of the acute coronary syndrome with proven therapy such as aspirin and β-blockers, rather than treating mild-to-moderate anaemia as an initial step. If the effects of RBC transfusion were either limited or increased then there would be no debate; however, the use of allogeneic RBCs has been shown to be associated with immunomodulation [12, 47] and/or alteration in the delivery of oxygen in the microcirculation [70, 71] , resulting in increased rates of infections and organ failure.

Few clinical studies have attempted to elucidate the risk : benefit ratio of anaemia and transfusion in cardiac patients. Two small RCTs [62, 72] examined transfusion practice in patients undergoing coronary artery bypass grafting, and concluded that a conservative approach to the administration of RBCs may be safe. However, two recent cohort studies suggested that anaemia may increase the risk of mortality in critical illness [73] and following surgery in patients with cardiovascular disease [74] . There were 418 and 420 patients in the restrictive and liberal transfusion groups, respectively. *Difference calculated by subtracting mean values of restrictive group from those of liberal group. † Three patients were lost to 60-day mortality rate; therefore n = 835. ‡ Nonsurvivors are considered to have all organs failing on date of death. § Changes in MOD score from baseline, while also incorporating adjustment for death. Data from Hébert et al [10] .

In a study of Jehovah's Witnesses (a group that refuses RBC transfusion on religious grounds) undergoing surgical procedures [74] , it was noted that mortality was significantly increased in patients with cardiac disease after a decrease in haemoglobin levels from 100-110 g/l to 60-69 g/l. In that study, patients with no cardiac disease and similar changes in haemoglobin levels showed no increase in mortality, which is in accordance with the results of the TRICC trial [10] . In the study by Hébert et al. [73] of 4470 critically ill patients, a correlation between Critical Care Vol 5 No 2 Alvarez et al [10] .

Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (all patients). Data from Hébert et al [10] .

Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (patients with APACHE II score ≤20). Data from Hébert et al [10] .

anaemia and mortality rates was observed. Those investigators also found that the risk of anaemia appeared to decrease with RBC transfusion in patients with cardiac disease. In patients with cardiac disease, mortality increased when haemoglobin concentrations were below 95 g/l, as compared with anaemic patients with other diagnoses (55% versus 42%; P = 0.09). In the subgroup of patients with cardiac disease, increasing haemoglobin values in anaemic patients was associated with improved survival (odds ratio 0.80 for each 10 g/l increase; P = 0.012). One possible explanation for the discrepancy between the TRICC trial and this observational study may be that the attending physicians who recruited patients into the study did not enter those patients who were considered to have severe cardiac disease.

Hébert et al. [73] sought to examine further whether a restrictive transfusion strategy was at least as effective as a liberal strategy in critically ill patients with cardiac disease. In the subgroup of patients with cardiovascular disease from the TRICC trial, those investigators suggested that most haemodynamically stable critically ill patients with cardiovascular disease may be transfused when haemoglobin concentrations fall below 70 g/l, and that the hemoglobin concentration should be maintained between 70 and 90 g/l. Average daily haemoglobin concentrations were 85 ± 6.2 g/l in the restrictive transfusion group and 103 ± 6.7 g/l in the liberal transfusion group (P < 0.01). In the 357 patients with cardiovascular disease, the 30-day mortality rate was 23% in the restrictive transfusion group versus 23% in the liberal group (95% confidence interval of the difference -8.4% to 9.1%; P = 1.00). Other mortality rates, including 60-day (26% versus 27%; P = 0.90), ICU (19% versus 16%; P = 0.49) and hospital mortality (27% versus 28%; P = 0.81), were not significantly different between groups. Kaplan-Meier survival curves comparing time to death demonstrated similar trends in the two groups ( Fig. 3 ; P = 0.98). The multiple organ dysfunction (MOD) scores, during the entire study period, were also not significantly different between groups (8.6 ± 4.9 versus 9.0 ± 4.4; P = 0.40), but the change in MOD score from baseline values was significantly lower in the restrictive group than in the liberal group (0.2 ± 4.2 versus 1.3 ± 4.4; P = 0.02).

Combined measures of morbidity and mortality, or composite outcomes, were also examined. When all patients who died were given a score of 24, the total MOD score between groups was not different (P = 0.39), or were the changes in MOD scores significantly different from baseline (2.7 ± 6.9 versus 4.0 ± 7.3; P = 0.08). Among the specific subset of cardiac patients with ischaemic heart disease (n = 257), there were no discernible differences in 30-day and 60-day as well as ICU mortality rates. However, a nonsignificant (P = 0.3) decrease in overall survival rate in the restrictive group was noted in those patients with confirmed ischaemic heart disease, severe peripheral vascular disease or severe comorbid cardiac disease (Fig. 4) .

In conclusion, a restrictive RBC transfusion strategy generally appears to be safe in most critically ill patients with cardiovascular disease, with the possible exception of patients experiencing acute myocardial infarction or unstable angina.

Survival over 30 days in patients with ischemic heart disease in the restrictive and liberal allogeneic RBC transfusion strategy groups. This graph illustrates Kaplan-Meier survival curves for all patients with ischemic heart disease in both study groups. There is no difference in mortality in patients in the restrictive group (dashed line) as compared to the liberal group (solid line) (P = 0.30).

The need to reduce the amount of allogeneic blood transfusions in order to reduce the associated risks has been firmly established. RBCs are associated with clinically important immune suppression, and stored RBCs have been shown to cause adverse microcirculatory effects that result in increased organ failure.

The question for some time has been whether critically ill patients are able to tolerate lower levels of haemoglobin without deleterious effects, thus reducing the amount of exposure to allogeneic transfusions. In the only large RCT, Hébert et al [10] established that there was no difference in mortality rates between restrictive and liberal transfusion strategies in noncardiac, critically ill patients. Although those investigators were able to show convincing trends that the liberal strategy may in fact be deleterious in terms of absolute values, statistical significance was not achieved. However, the fact that no difference between the two strategies was achieved is of great importance, because this means that the total number of transfusions can be reduced by approximately half without any impact on mortality. In addition, these findings are easily put into clinical practice. Although many questions remain, the TRICC trial and many laboratory and clinical studies have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. As such, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of RBCs, save blood overall, and decrease health care costs. The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 The 21st International Symposium on Intensive Care and Emergency Medicine was dominated by the results of recent clinical trials in sepsis and acute respiratory distress syndrome (ARDS). The promise of extracorporeal liver replacement therapy and noninvasive ventilation were other areas of interest. Ethical issues also received attention. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of a high standard. The meeting was marked by a sense of renewed enthusiasm that positive progress is occurring in intensive care medicine. This year's symposium was dominated by the results of recent clinical trials. After 10 years of 'magic bullet' trials in sepsis, a number of successful therapeutic options are now emerging. In addition, recent advances in our understanding of the soup of mediators observed in sepsis offer yet more tantalizing targets for new therapies.

In contrast, the eagerly awaited results from Italy of the prone positioning trial in ARDS were disheartening. The epidemiology of both sepsis and ARDS, and their impact on clinical studies and the future provision of critical care were also hot topics. The era of extracorporeal liver replacement therapy is upon us, with considerable early promise and the probability of wide availability. Finally, as always, ethics remained an area of interest.

This report summarizes and discusses the presentations on the above topics.

Angus (Pittsburgh, PA, USA) presented his group's work on the epidemiology of sepsis in the USA (accepted for publication in Critical Care Medicine). They developed a method for identifying hospitalized patients with sepsis based on ICD9 criteria, the most widely recorded coding system used in US hospitals. Prospective testing of the method found it to be both sensitive and reliable. They then applied it to a representative selection of US hospitals. Their results indicated that about 50% of intensive care unit (ICU) patients have systemic inflammatory response syndrome, and that approximately 20% of these progress to severe sepsis. Mortality for severe sepsis was greater than 30%. Demographically, those at the extremes of age represent the most at-risk groups, in whom the mortality is also the highest. These data provides yet another reminder that the increasing demands on health care resources caused by the ageing population is predicted to exceed intensive care provision within the next The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 10-20 years. Finally, those investigators found a striking demographic peak in patients aged 20-30 years, which they attributed largely to human immunodeficiency virus.

The long-standing debate between the two schools of sepsis theory -microcirculatory dys-autoregulation versus cellular dysfunction -shows signs of resolution. New techniques for studying tissue oxygen tension, presented by Ince (Amsterdam, The Netherlands), provide more evidence that microcirculatory dys-autoregulation results in significant shunting. This occurs predominantly in the submucosal and serosal portions of organs, and is an early event. These studies show that the macroscopic restoration of global oxygen delivery fails to improve oxygen consumption as the mucosa becomes hyperoxic, whereas the submucosa and serosa remain hypoxic. Somewhat counterintuitively, this can be reversed in the face of resistant hypotension with vasodilators, at least in animal models.

The cellular dysfunction camp, although still somewhat doubtful as to the importance of these microcirculatory findings, have now clearly established that their championed mechanism of mitochondrial failure is a late but crucial event in the evolution of sepsis. Fink (Pittsburgh, PA, USA) presented evidence that mitochondrial failure in septic cells is triggered by the activation of the enzyme poly-adenosine diphosphate ribose polymerase [1] . This enzyme represents a significant target for novel therapies, which are apparently already in development. The debate regarding the toxicity of oxygen and the formation of free radicals continues despite the absence of demonstrated effectiveness of scavenging therapies, and is a testament to the incomplete understanding of this area.

The round-table conference preceding this year's symposium concentrated on distilling current knowledge on the microscopic events in critically ill patients into an explanation of the macroscopic multiorgan failure that is so commonly encountered. The conclusions of the conference appeared to relate mostly to future directions for research, in particular the study of organ-organ interactions. Marshall (Toronto, Canada) proposed the development of an alternative to the much-maligned physiological scoring systems, based on the staging systems widely used in the field of oncology. He proposed that mediator levels, in addition to physiological variables, will soon be used usefully to characterize septic patients. He also suggested that, in the light of the recent successful mediator trials in sepsis, future therapies will be directed in a manner analogous to the control of glucose in diabetic patients.

The natural anticoagulants antithrombin III (AT III), tissue factor pathway inhibitor (TFPI) and activated protein C (APC), and the cytokine tumour necrosis factor (TNF)-α are the latest inflammatory mediators to be targeted in large multicentre clinical trials in an attempt to improve the current dismal outcome for patients with severe sepsis.

The KyberSept AT III study recruited over 2300 patients from 200 centres, with high Simplified Acute Physiology Scale scores (median 50), and a mortality of nearly 40% [2] . Unfortunately, no overall benefit was shown between AT III and placebo, although results were more encouraging in an analysis of the subgroup of patients who received AT III but no heparin, which is known to inhibit AT III. Interestingly, improvements in quality of life scores were seen in survivors who received AT III in comparison to those who received placebo, suggesting that morbidity may be reduced, although again this was an analysis of a subgroup. Patients in the AT III group who received concomitant heparin had a significantly higher incidence of bleeding events, and outcome worsened as the dose of heparin increased. Explanations for the failure of this study included the inhibitory effects of heparin and the failure to achieve AT III activity levels of greater that 200% from baseline in the treatment population, a level established as required for therapeutic benefit in phase II trials.

Phase II clinical trial results using TFPI (TFPI n = 141, placebo n = 69; unpublished data) show a mortality benefit in the sicker sepsis patients who already have coagulation problems. Results of the phase III multicentre study are expected to be presented at the 22nd International Symposium on Intensive Care and Emergency Medicine, in Brussels in 2002.

Human trials of various anti-TNF-α formulations have been variable to date, and include North American sepsis trial (NORASEPT) I [3] , International sepsis trial (INTERSEPT) [4] and NORASEPT II [5] . Possible reasons have included a lack of biological activity of the anti-TNF-α formulation studied, inappropriate timing of therapy, redundancy of proinflammatory mediators and hetereogeneity of patient populations. The Monoclonal Anti-TNF, A Randomized controlled Sepsis Trial (MONARCS) study used a different anti-TNF-α formulation (F[ab′]2 fragment of a murine monoclonal antibody to human TNF-α), and stratified patients based on demonstrable abnormalities in immunological pathways (highly elevated interleukin-6 levels -a circulating cytokine that is induced by TNF-α). Unpublished results revealed 28-day mortality rates of 44 and 48% in the anti-TNF-α and placebo groups, respectively, in those patients who had high interleukin-6 levels on recruitment to the study (n = 488 anti-TNF-α, n = 510 placebo). This represented a relative mortality reduction of 14%. Relative mortality reduction in all patients (n = 1305 anti-TNF-α, n = 1329 placebo), independent of baseline interleukin-6 levels, was only 10%.

The Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study is hot off the press [6] , and presentation of the results at the congress allowed those of us who still carry the unopened New England Journal of Medicine issue in our briefcases to catch up! A total of 164 sites from 11 countries recruited 1690 patients with severe sepsis, before the trial was prematurely stopped following the second safety analysis. Twenty-eight-day all-cause mortality rates for placebo and APC were 31 and 25% respectively, with a relative risk reduction of 19%. Resolution of cardiovascular and respiratory function was more rapid in survivors who received APC, although ICU and hospital stay did not differ. There was a trend towards an increase in serious bleeding events in the APC group (3% APC versus 2% placebo), but these events were primarily due to trauma or instrumentation. Although this is an exciting breakthrough, we all recognize that when APC reaches the market place it will seriously stretch ICU finances, especially because there appear to be other mediators on the horizon that we will be encouraged to use, in combination, to fight the inflammatory 'soup'.

Two opposing epidemiological views of ARDS were presented by Lemaire (Créteil, France) and Evans (London, UK). Broad agreement does seem to exist as to the incidence of this condition, which is in the order of 10/100,000, although there is significant variation between countries. It was argued that this variation results from the availability of ventilated beds, with higher incidences apparent in countries with greater provision, emphasizing that this condition can be considered the result of intensive care intervention or, as one speaker put it, 'physician-induced lung injury'.

Early results from the Acute Lung Injury Verification of European Epidemiology (ALIVE) study (unpublished data), sponsored by the European Society of Intensive Care Medicine, are at odds with recent trial findings. The ALIVE study, which included over 6000 patients surveyed in 1998, found a 50-60% 28-day mortality, which compares to only 20-30% in the control groups of recent trials. Pneumonia was the commonest cause, responsible for 50% of cases, with sepsis identified as the cause in a further 20-30%. Astonishingly, this study found the ratio of arterial oxygen tension to fractional inspired oxygen at ICU admission was highly predictive of mortality, despite continuing controversy regarding this measurement.

A diverse range of views were presented from the Third International Consensus Conference on ARDS (unpublished data), held in Barcelona late last year. The decision as to how to change the defining criteria for this condition remains unresolved. The debates surrounding chest X-ray criteria, the use of the ratio of arterial oxygen tension to fractional inspired oxygen, and the level/utility of pulmonary artery wedge pressure measurements continue.

In addition, a debate has arisen as to whether ARDS can be a unilateral process, and whether it can coexist with cardiac failure. There appears to be increasing recognition that ARDS represents only a small subset of patients with acute lung failure (approximately 30%). Surprisingly little is known about the remainder of this larger group. In contrast to the ALIVE study, several centres have reported their 28-day mortality at 40%, which represents an improvement from the 60% of 10 years ago. However, it was argued that a 28-day follow-up period is too short for clinical trials, as the long-term quality of life for patients with ARDS is poor compared with that of critically ill patients without this condition. Results suggest that the recovery of lung function is good overall, but is dependent on severity. Treatment recommendations include the universal adoption of the US National Institutes of Health protective lung ventilation strategy [7] . There was general agreement that recruitment manoeuvres are beneficial, but how and when to employ them remains controversial.

Rouby (Paris, France) put forward a new classification for ARDS based on computed tomography findings. He observed that patients can be split into three groups, depending on the appearance of the upper lobes. In group 1 the upper lobes are normal, and positive end-expiratory pressure (PEEP) is of little benefit and results in significant over-distension. Survival in this group is approximately 60%. In group 2 the upper lobes are abnormal, PEEP is of dramatic benefit, but survival is only approximately 25%. In group 3 there are mixed/patchy abnormalities, the effects of PEEP are less predictable, but, as in group 1, survival is approximately 60%.

Gattinoni (Milan, Italy) presented the results of the longawaited Italian multicentre randomized controlled trial of prone positioning in ARDS (unpublished data). This trial was terminated after 3 years despite having only recruited 304 patients; enrollment of 750 patients was originally planned, in order to achieve sufficient statistical power. At trial outset, recruitment was encumbered by the lack of familiarity with and scepticism regarding this procedure in many of the centres. However, by the end of the study many participants were unwilling to enter patients into the trial, because they felt it unethical to deny them this intervention. The trial protocol resulted in patients in the treatment group being prone for an average of only 7 out of 24 h for a 10-day period. Overall there was no difference in mortality between the control and treatment groups at day 10, time of ICU discharge, or at 6 months. Interestingly, analysis of subgroups revealed a significant difference in the outcome at 10 days for patients with the most severe disease, although this disappeared by ICU discharge. In retrospect, the design of this ambitious trial was flawed by its failure to establish the optimal utilization of this manoeuvre.

The opening session reported that we are making progress in supporting the failing liver (Wendon, London, UK). Current optimism should probably be limited to extracorporeal methods, because the molecular adsorbent recirculating system (essentially extracorporeal albumin dialysis) has been shown to have beneficial clinical effects as well as improved survival in two small randomized controlled trials [8, 9] . The equipment is familiar to all those who use dialytic therapies, and we will undoubtedly hear more about this system in the next few years.

The slide of a patient reading the newspaper through a transparent helmet, while receiving noninvasive ventilation (NIV) resembled pictures of a NASA astronaut! However, it was reported to be well tolerated for prolonged periods, and significantly reduces the complications associated with NIV (pressure areas, tolerance of mask). The recent Consensus Conference [10] examined weaning aspects of NIV and emphasized the reduced weaning time and avoidance of reintubation, but called for more randomized controlled trials. Finally, although continuous positive airway pressure has been shown to be beneficial in pulmonary oedema, caution is still advised with the use of bilevel positive airway pressure because of the reporting of myocardial infarction in several studies. However, the groups studied were unmatched and starting points were different, so conclusions should not be drawn until randomized controlled trial results are available in this area.

This was a well-attended session, which, according to Levy (Providence, RI, USA), was in complete contrast to the interest shown in the USA for the subject. Although there were few new data in the session, the emphasis on a strategy for lawsuits was welcome. Suggestions included statements from scientific societies at a national and international level, open reporting in medical files of decisions to withdraw or withhold treatment, and family involvement in decision making that will ultimately involve better media education.

The last day of this year's symposium was sadly abandoned by many due to the Belgian rail strike. Despite this, the usual convivial atmosphere, both in and around the congress, was as abundant as ever. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of the usual high standard, although, yet again, access to many of the symposium's venues was limited by the lack of capacity of the secondary rooms. The 21st International Symposium was marked by a sense of renewed enthusiasm that real positive progress is occurring at the coal face of intensive care. Heme oxygenase-1 and carbon monoxide in pulmonary medicine Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states. The heme oxygenase-1/carbon monoxide (HO-1/CO) system has recently seen an explosion of research interest due to its newly discovered physiological effects. This metabolic pathway, first characterized by Tenhunen et al. [1, 2] , has only recently revealed its surprising cytoprotective properties [3, 4] . Research in HO-1/CO now embraces the entire field of medicine where reactive oxygen/nitrogen species, inflammation, growth control, and apoptosis represent important pathophysiological mechanisms [3] [4] [5] [6] . Indeed, the number of publications in recent years concerning HO-1 has increased exponentially, while the list of diseases and physiological responses associated with changes in HO-1 continues to expand [5] .

Until now, relatively few studies have addressed the role of HO-1/CO in pulmonary medicine. Several investigators have focused on the diagnostic application of the HO-1/CO system, by measuring exhaled CO (E-CO) in various pathological pulmonary conditions, such as asthma or chronic obstructive pulmonary disease (COPD) [7] . In another experimental approach, investigators have examined the expression of HO-1 in lung tissue from healthy or diseased subjects [8, 9] . This review will highlight the actions of HO-1/CO in the context of heme degradation have antioxidant properties [18, 19] . The liberated heme iron undergoes detoxification either by extracellular efflux or by sequestration into ferritin, an intracellular iron-storage molecule with potential cytoprotective function [20] [21] [22] [23] . Of the three known isoforms of HO (HO-1, HO-2, and HO-3), only HO-1 responds to xenobiotic induction [24] [25] [26] [27] . Constitutively expressed in many tissues, HO-2 occurs at high levels in nervous and vascular tissues, and may respond to regulation by glucocorticoids [25, 28, 29] . HO-1 and HO-2 differ in genetic origin, in primary structure, in molecular weight, and in their substrate and kinetic parameters [25, 26] . HO-3 displays a high sequence homology with HO-2 but has little enzymatic activity [27] . This review will focus on the inducible, HO-1, form.

In addition to the physiological substrate heme, HO-1 responds to induction by a wide variety of stimuli associated with oxidative stress. Such inducing agents include hypoxia, hyperoxia, cytokines, nitric oxide (NO), heavy metals, ultraviolet-A (320-380 nm) radiation, heat shock, shear stress, hydrogen peroxide, and thiol (-SH)-reactive substances [3] . The multiplicity of toxic inducers suggest that HO-1 may function as a critical cytoprotective molecule [3, 4] . Many studies have suggested that HO-1 acts as an inducible defense against oxidative stress, in models of inflammation, ischemia-reperfusion, hypoxia, and hyperoxia-mediated injury (reviewed in [3] ). The mechanisms by which HO-1 can mediate cytoprotection are still poorly understood. All three products of the HO reaction potentially participate in cellular defense, of which the gaseous molecule CO has recently received the most attention [30, 31] . The administration of CO at low concentrations can compensate for the protective effects of HO-1 in the presence of competitive inhibitors of HO-1 activity [32] [33] [34] . While HO-1 gene transfer confers protection against oxidative stress in a number of systems, clearly not all studies support a beneficial role for HO-1 expression. Cell-culture studies have suggested that the protective effects of HO-1 overexpression fall within a critical range, such that the excess production of HO-1 or HO-2 may be counterprotective due to a transient excess of reactive iron generated during active heme metabolism [35, 36] . Thus, an important caveat of comparative studies on the therapeutic effects of CO administration versus HO-1 gene delivery arises from the fact that the latter approach, in addition to producing CO, may have profound effects on intracellular iron metabolism.

HO-1 expression is primarily regulated at the transcriptional level. Genetic analyses have revealed two enhancer sequences (E1, E2) in the murine HO-1 gene located at -4 kb (E1) and -10 kbp (E2) of the transcriptional start site [37, 38] . These enhancers mediate the induction of HO-1 by many agents, including heavy metals, phorbol esters, endotoxin, oxidants, and heme. E1 and E2 contain repeated stress-responsive elements, which consist of overlapping binding sites for transcription factors including activator protein-1 (AP-1), v-Maf oncoprotein, and the cap'n'collar/basic-leucine zipper family of proteins (CNC-bZIP), of which Nrf2 (NF-E2-related factor) may play a critical role in HO-1 transcription [39] . The promoter region of HO-1 also contains potential binding sites for nuclear factor κB (NF-κB), though the functional significance of these are not clear [40] . Both NF-κB and AP-1 have been identified as regulatory elements responsive to oxidative cellular stress [40, 41] . In response to hyperoxic stress, AP-1 factors mediated the induction of HO-1 in cooperation with signal-transducer and activator of transcription (STAT) proteins [41] . Furthermore, a distinct hypoxia-response element (HRE), which mediates the HO-1 response to hypoxia, represents a binding site for the hypoxia-inducible factor-1 (HIF-1) [42] .

The toxic properties of CO are well known in the field of pulmonary medicine. This invisible, odorless gas still claims many victims each year by accidental exposure. CO evolves from the combustion of organic materials and is present in smoke and automobile exhaust. The toxic actions of CO relate to its high affinity for hemoglobin (240-fold greater than that of O 2 ). CO replaces O 2 rapidly from hemoglobin, causing tissue hypoxia [43] [44] [45] . At high concentrations, other mechanisms of CO-induced toxicity may include apoptosis, lipid peroxidation, and inhibition of drug metabolism and respiratory enzyme functions [44] .

Only recently has it become known that, at very low concentrations, CO participates in many physiological reactions. Where a CO exposure of 10,000 parts per million (ppm) (1% by volume CO in air) is toxic, 100-250 ppm (one hundredth to one fortieth as much) will stimulate the physiological effects without apparent toxicity [4] . The majority of endogenous CO production originates from active heme metabolism (>86%), though a portion may be produced in lipid peroxidation and drug metabolism reactions [46] . Cigarette smoking, still practiced by many lung patients, represents a major source of chronic lowlevel exposure to CO. Inhaled CO initially targets alveolar macrophages and respiratory epithelial cells.

The exact mechanisms by which CO acts at the molecular level remain incompletely understood. CO potentially exerts its physiological effects by influencing at least three known pathways (Fig. 2 ). By complexation with the heme moiety of the enzyme, CO activates soluble guanylate cyclase (sGC), stimulating the production of cyclic 3':5'guanosine monophosphate (cGMP) [47] . The sGC/cGMP pathway mediates the effects of CO on vascular relaxation, smooth muscle cell relaxation, bronchodilation, neurotransmission, and the inhibition of platelet aggregation, coagulation, and smooth muscle proliferation [48] [49] [50] [51] . Furthermore, CO may cause vascular relaxation by directly activating calcium-dependent potassium channels [52] [53] [54] . CO potentially influences other intracellular signal transduction pathways. The mitogen-activated protein kinase (MAPK) pathways, which transduce oxidative stress and inflammatory signaling (i.e. response to lipopolysaccharide), may represent an important target Possible mechanism(s) of carbon monoxide action Figure 2 Possible mechanism(s) of carbon monoxide action. Endogenous carbon monoxide (CO) arises principally as a product of heme metabolism, from the action of heme oxygenase enzymes, although a portion may arise from environmental sources such as pharmacological administration or accidental exposure, or other endogenous processes such as drug and lipid metabolism. The vasoregulatory properties of CO, including its effects on cellular proliferation, platelet aggregation, and vasodilation, have been largely ascribed to the stimulation of guanylate cyclase by direct heme binding, leading to the generation of cyclic GMP. The anti-inflammatory properties of CO are associated with the downregulation of proinflammatory cytokine production, dependent on the selective modulation of mitogen-activated protein kinase (MAPK), such as the 38 kilodalton protein (p38MAPK). In addition to these two mechanisms, CO may potentially interact with any hemoprotein target, though the functional consequences of these interactions with respect to cellular signaling remain poorly understood.

Anti-Platelet Aggregation Anti-Proliferation

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Inhibition of pro-inflammatory cytokine production

Modulation of hemoprotein function of CO action [32, 34, 55, 56 ]. An anti-apoptotic effect of CO and its relation to MAPK has recently been described. The overexpression of HO-1 or the exogenous administration of CO prevented tumor necrosis factor α (TNF-α)induced apoptosis in murine fibroblasts [57] . In endothelial cells, the anti-apoptotic effect of CO depended on the modulation of the p38 (38 kilodalton protein) MAPK pathway [34] . The role of the remaining heme metabolites, (i.e. Fe and biliverdin IXα) in the modulation of apoptosis is currently being investigated and is beyond the scope of this review. Recent studies have reported a potent anti-inflammatory effect of CO, involving the inhibition of proinflammatory cytokine production after endotoxin stimulation, dependent on the modulation of p38 MAPK [32] . The clinical relevance of p38 MAPK lies in the possibility of modulating this pathway in various clinical conditions to downregulate the inflammatory response [58] .

Oxidative stress arising from an imbalance between oxidants and antioxidants plays a central role in the pathogenesis of airway disease [59] . In lung tissue, HO-1 expression may occur in respiratory epithelial cells, fibroblasts, endothelial cells, and to a large extent in alveolar macrophages [41, 60, 61] . HO-1 induction in these tissues, in vitro and in vivo, responds to common causes of oxidative stress to the airways, including hyperoxia, hypoxia, endotoxemia, heavy metal exposure, bleomycin, diesel exhaust particles, and allergen exposure [4, 41, 61] . Induction of HO-1 or administration of CO can protect cells from these stressful stimuli [10, 41] . In one of the experiments that best illustrate the protective role of CO in vivo, rats were exposed to hyperoxia (>98% O 2 ) in the absence or presence of CO at low concentration (250 ppm). The CO-treated rats showed increased survival and a diminished inflammatory response to the hyperoxia [11] . As demonstrated in a model of endotoxin-induced inflammation, the protection afforded by CO most likely resulted from the downregulated synthesis of proinflammatory cytokines (i.e. TNF-α, IL-1β) and the upregulation of the anti-inflammatory cytokine interleukin-10 (IL-10) [32] . Furthermore, increases in exhaled CO (E-CO) have been reported in a number of pathological pulmonary conditions, such as unstable asthma, COPD, and infectious lung disease; these increases may reflect increased endogenous HO-1 activity [7] . Elevated carboxyhemoglobin (Hb-CO) levels have also been reported in these same diseases in nonsmoking subjects, where both the E-CO and Hb-CO levels decrease to normal levels in response to therapy [62] .

E-CO in humans originates primarily from both systemic heme metabolism, which produces CO in various tissues, and localized (lung) heme metabolism, as a result of the combined action of inducible HO-1 and constitutive HO-2 enzymatic activity. Endogenously produced or inspired CO is eliminated exclusively by respiration [63] . Elevation of E-CO may also reflect an increase in exogenous sources such as smoking or air pollution. In addition to changes in environmental factors, elevations of E-CO in lung diseases may reflect an increase in blood Hb-CO levels in response to systemic inflammation, as well as an increase in pulmonary HO-1 expression in response to local inflammation [9, 62, 64] .

The diagnostic value of measuring E-CO remains controversial due to many conflicting reports (i.e. some reports indicate differences in E-CO measurements between disease activity and controls, and some reports do not). The possible explanations for these discrepancies include large differences in patient populations and in the methods used for measuring E-CO, and undefined corrections for background levels of CO. Furthermore, remarkable differences arise between studies in the magnitude of the E-CO levels in the control groups as well as in treated or untreated asthma patients. When active or passive smoking occurs, or in the presence of high background levels of CO, the measurement of E-CO is not particularly useful for monitoring airway inflammation. In patients who smoke, E-CO can be used only to confirm the smoking habit [65, 66] . Comparable to the beginning era of measurements of exhaled NO, a standardization in techniques and agreement on background correction should be reached for E-CO measurements, to allow proper conclusions to be drawn in this area of investigation.

Asthma, a form of allergic lung disease, features an accumulation of inflammatory cells and mucus in the airways, associated with bronchoconstriction and a generalized airflow limitation. Inflammation, a key component of asthma, involves multiple cells and mediators where an imbalance in oxidants/antioxidants contributes to cell damage. Several pathways associated with oxidative stress may participate in asthma. For example, the redox-sensitive transcription factors NF-κB and AP-1 control the expression of proinflammatory mediators [59, [67] [68] [69] .

In light of the potential protective effects of HO-1/CO on inflammatory processes, the study of HO-1 in asthma has gained popularity. In a mouse model of asthma, HO-1 expression increased in lung tissue in response to ovalbumin aerosol challenge, indicating a role for HO-1 in asthma [70] . In a similar model of aeroallergen-induced asthma in ovalbumin-sensitized mice, exposure to a CO atmosphere resulted in a marked attenuation of eosinophil content in bronchoalveolar lavage fluid (BALF) and downregulation of the proinflammatory cytokine IL-5 [10] . This experiment showed that exogenous CO can inhibit asthmatic responses to allergens in mice.

Recent human studies have revealed higher HO-1 expression in the alveolar macrophages and higher E-CO in untreated asthmatic patients than in healthy nonsmoking controls [71, 72] . Patients with exacerbations of asthma and patients who were withdrawn from inhaled steroids showed higher E-CO levels than steroid-treated asthmatics or healthy controls [73] . Higher levels of E-CO may also occur in children with persistent asthma than in healthy controls [74] . E-CO levels may correlate with functional parameters such as peak expiratory flow rate. A low rate in asthma exacerbations correlated with high E-CO, whereas normalization of the rate with oral glucocorticoid treatment resulted in a reduction of E-CO [75] . Furthermore, increased E-CO was associated with greater expression of HO-1 in airway alveolar macrophages obtained by induced sputum in untreated asthmatic patients than in controls. These asthma patients also showed higher bilirubin levels in the induced sputum, indicating higher HO activity [71] . Furthermore, patients with asthma show an increased Hb-CO level at the time of exacerbation, with values decreasing to control levels after oral glucocorticoid treatment [62] . In human asthmatics, E-CO and airway eosinophil counts decreased in response to a one-month treatment with inhaled corticosteroids [73] . In direct contrast to such studies promoting E-CO as a useful noninvasive tool for monitoring airway inflammation, other studies reported no difference in E-CO levels of asthma patients versus healthy controls, or between patients with stable and unstable asthma. In one such report, no further change in E-CO occurred in asthma patients after a one-month treatment of inhaled corticosteroids, despite observed decreases in airway eosinophil content and bronchial responsiveness to metacholine [76] . A recent study accentuates this finding in asthma excerbations, where no decrease in E-CO of children with asthma could be detected after oral prednisolone treatment [77] . In human allergic responses, results on elevation of E-CO are also inconclusive. A clear elevation of E-CO after allergen exposure occurred in patients with asthma during the late response, and during the early response immediately after the inhalation [78] . However, another report showed that no elevation of E-CO occurred in allergen-induced asthma within 48 hours after allergen challenge [79] . Finally, increases in E-CO were measured in allergic rhinitis, correlating with seasonal changes in exposure to allergen (pollen) [80] .

Airway inflammation plays an important role in the development of COPD, characterized by the presence of macrophages, neutrophils, and inflammatory mediators such as proteinases, oxidants, and cytokines. Further-more, the inflammatory consequences of chronic microbiological infections may contribute to the progression of the disease. The current paradigm for the pathogenesis of COPD involves imbalances in protease/antiprotease activities and antioxidant/pro-oxidant status. Proteases with tissue-degrading capacity, (i.e. elastases and matrix metalloproteinases), when insufficiently inhibited by antiproteases, can induce tissue damage leading to emphysema. Oxidants that supersede cellular antioxidant defenses can furthermore inactivate antiproteases, cause direct injury to lung tissue, and interfere with the repair of the extracellular matrix. Smoking plays an important role in both hypotheses. Cigarette smoke will act primarily on alveolar macrophages and epithelial cells, which react to this oxidative stress by producing proinflammatory cytokines and chemokines and releasing growth factors. Nevertheless, smoking cannot be the only factor in the development of COPD, since only 15-20% of smokers develop the disease [81, 82] .

Exposure to reactive oxygen species (from cigarette smoke or chronic infections) and an imbalance in oxidant/antioxidant status are the main risk factors for the development of COPD. To defend against oxidative stress, cells and tissues contain endogenous antioxidant defense systems, which include millimolar concentrations of the tripeptide glutathione (GSH). A close relation exists between GSH concentration and HO-1, whereby depletion of GSH augments the transcriptional regulation of HO-1 by oxidants, suggesting that the HO-1/CO system acts as a secondary defense against oxidative stress [83] [84] [85] [86] . Accumulating clinical evidence suggests that HO-1/ CO may also play an important part in COPD. Alveolar macrophages, which produce a strong HO-1 response to stimuli, may represent the main source of CO production in the airways [60, 64] . Patients with COPD have displayed higher E-CO than healthy nonsmoking controls [87] . Furthermore, much higher levels of HO-1 have been observed in the airways of smokers than in nonsmokers [64] . Among subjects who formerly smoked, patients with COPD have lower HO-1 expression in alveolar macrophages than healthy subjects [88] . A microsatellite polymorphism that is linked with the development of COPD may occur in the promoter region of HO-1, resulting in a lower production of HO-1 in people who have the polymorphism. Thus, a genetically dependent downregulation of HO-1 expression may arise in subpopulations, possibly linked to increased susceptibility to oxidative stress [89] [90] [91] . Future studies on both genetic predisposition and possible therapeutic modalities will reveal the involvement of the HO-1/CO system in COPD.

Cystic fibrosis (CF) involves a deposition of hyperviscous mucus in the airways associated with pulmonary dysfunc-tion and pancreatic insufficiency, which may be accompanied by chronic microbiological infections. E-CO readings were higher in untreated versus oral-steroidtreated CF patients [92] . Furthermore, E-CO increased in patients during exacerbations of CF, correlating to deterioration of the forced expiratory volume in one second (FEV 1 ), with normalization of the E-CO levels after treatment [93] . E-CO levels may correlate with exhaled ethane, a product of lipid peroxidation that serves as an indirect marker of oxidative stress. Both E-CO and exhaled ethane were higher in steroid-treated and untreated CF patients than in healthy controls [94] . E-CO was higher in children with CF than in control patients. In addition to the inflammatory and oxidative stress responses to continuous infectious pressure in these patients, E-CO may possibly respond to hypoxia. E-CO increased further in CF children following an exercise test, and correlated with the degree of oxyhemoglobin desaturation, a finding suggestive of an increased HO-1 expression in CF patients during hypoxic states induced by exercise [95] .

In patients with pneumonia, higher Hb-CO levels can be measured at the onset of illness, with values decreasing to control levels after antibiotic treatment [62] . E-CO levels were reported to be higher in lower-respiratory-tract infections and bronchiectasis, with normalization after antibiotic treatment [96, 97] . Furthermore, E-CO levels in upper-respiratory-tract infections were higher than in healthy controls [74, 80] . The relationship between higher measured E-CO in these infectious states and higher Hb-CO levels cannot be concluded from these studies.

The role of HO-1 in the development of interstitial lung disease remains undetermined. Comparative immunohistochemical analysis has revealed that lung tissue of control subjects, patients with sarcoidosis, usual interstitial pneumonia, and desquamative interstitial pneumonia, all showed a high expression of HO-1 in the alveolar macrophages but a weak expression in the fibrotic areas [98] . The antiproliferative properties of HO-1 suggest a possible beneficial role in limiting fibrosis; however, this hypothesis is complicated by a newly discovered relation between IL-10 and HO-1. IL-10 produced by bronchial epithelial cells promotes the growth and proliferation of lung fibroblasts [99] . HO-1 expression and CO treatment have been shown to increase the production of IL-10 in macrophages following proinflammatory stimuli [32] . Conversely, IL-10 induces HO-1 production, which is apparently required for the anti-inflammatory action of IL-10 [100] .

A recent report clearly shows the suppression of bleomycin-induced pulmonary fibrosis by adenovirus-mediated HO-1 gene transfer and overexpression in C57BL/6 mice, involving the inhibition of apoptotic cell death [101] .

Overall, more research is needed to elucidate the mechanisms of HO-1 in interstitial lung disease and its possible therapeutic implications.

HO-1 action may be of great importance in solid tumors, an environment that fosters hypoxia, oxidative stress, and neovascularization. HO-1 may have both pro-and antagonistic effects on tumor growth and survival. HO-1 and CO cause growth arrest in cell-culture systems and thus may represent a potential therapeutic modality in modulating tumor growth [16] . The overexpression of HO-1 or administration of CO in mesothelioma and adenocarcinoma mouse models resulted in improved survival (>90%) as well as reduction in tumor size (>50%) [17] . Furthermore, HO-1 expression in oral squamous cell carcinomas can be useful in identifying patients at low risk of lymph node metastasis. High expression of HO-1 was detected in groups without lymph node metastasis in this report [102] . In contrast to growth arrest, HO-1 may protect solid tumors from oxidative stress and hypoxia, possibly by promoting neovascularization. In one study, zinc protoporphyrin, a competitive inhibitor of HO-1 enzyme activity, suppressed tumor growth [103] .

CO may represent a critical mediator of the body's adaptive response to hypoxia, a common feature in pulmonary vascular disease [104] . Since CO can modulate vascular tone by inducing cGMP and large, calcium-dependent potassium channels, HO-1 and CO probably play important roles in pulmonary vascular diseases [54] . A NOmediated HO-1 induction occurred in the hepatopulmonary syndrome during cirrhosis, associated with enhancement of vascular relaxation [105] . In portopulmonary hypertension, elevated levels of cGMP and inducible nitric oxide synthase (iNOS) expression in the vascular endothelium, and HO-1 expression in macrophages and bronchial epithelium have been described [106] . In transgenic mice models, ho-1 -/and ho-1 +/+ mice did not differ in their development of pulmonary hypertension following chronic hypoxia treatment, despite the development of right ventricular dilation and right myocardial infarction in ho-1 -/mice [107] . The preinduction of HO-1 protein with chemical inducers, however, prevented the development of pulmonary hypertension in the rat lung as a consequence of chronic hypoxia treatment [108] . Transgenic mice overexpressing HO-1 in the lung were resistant to hypoxia-induced inflammation and hypertension [109] . Further research is needed to elucidate the potential role of HO-1 and CO in primary human lung vascular diseases such as primary pulmonary hypertension.

Supplemental oxygen therapy is often used clinically in the treatment of respiratory failure. Exposure to high oxygen tension (hyperoxia) may cause acute and chronic lung injury, by inducing an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema [110, 111] . Hyperoxia-induced lung injury causes symptoms in rodents that resemble human acute respiratory distress syndrome [112] .

Hyperoxia induced HO-1 expression in adult rats but apparently not in neonatal rats, in which the expression and activities of HO-1 and HO-2 are developmentally upregulated during the prenatal and early postnatal period [113] .

Both HO-1 and HO-2 potentially influence pulmonary adaptation to high O 2 levels. In one example, the adenoviral-mediated gene transfer of HO-1 into rat lungs protected against the development of lung apoptosis and inflammation during hyperoxia [114] . In vitro studies showed that the overexpression of HO-1 in lung epithelial cells or rat fetal lung cells caused growth arrest and conferred resistance against hyperoxia-induced cell death [15, 16] . An oxygen-tolerant variant of hamster fibroblasts that moderately overexpressed HO-1 in comparison with the parent line resisted oxygen toxicity in vitro. The treatment of this oxygen-tolerant strain with HO-1 antisense oligonucleotides reduced the resistance to hyperoxia. In contrast, additional, vector-mediated, HO-1 expression did not further increase oxygen tolerance in this model [115] .

In vivo studies with gene-deleted mouse strains have provided much information on the roles of HO-1 and HO-2 in oxygen tolerance. Dennery et al. demonstrated that heme oxygenase-2 knockout mice (ho-2 -/-) were more sensitive to the lethal effects of hyperoxia than wild-type mice [116] . In addition to the absence of HO-2 expression, however, the mice displayed a compensatory increase in HO-1 protein expression, and higher total lung HO activity. Thus, in this model, the combination of HO-2 deletion and HO-1 overexpression resulted in a hyperoxiasensitive phenotype. Recent studies of Dennery et al. have shown that HO-1-deleted (ho-1 -/-) mice were more resistant to the lethal effects of hyperoxia than the corresponding wild type [117] . The hyperoxia resistance observed in the ho-1 -/strain could be reversed by the reintroduction of HO-1 by adenoviral-mediated gene transfer [117] . In contrast, mouse embryo fibroblasts derived from ho-1 -/mice showed increased sensitivity to the toxic effects of hemin and H 2 O 2 and generated more intracellular reactive oxygen species in response to these agents [118] . Both ho-1 -/-and ho-2 -/strains were anemic, yet displayed abnormal accumulations of tissue iron. Specifically, ho-1 -/accumulated nonheme iron in the kidney and liver and had decreased total iron content in the lung, while ho-2 -/mice accumulated total lung iron in the absence of a compensatory increase in ferritin levels [116, 119] . The mechanism(s) by which HO-1 or HO-2 deletions result in accumulation of tissue iron remain unclear. These studies, taken together, have indicated that animals deficient in either HO-1 and HO-2 display altered sensitivity to oxidative stress conditions. Aberrations in the distribution of intra-and extra-cellular iron, may underlie in part, the differential sensitivity observed [116, 117] .

Otterbein et al. have shown that exogenous CO, through anti-inflammatory action, may protect the lung in a rat model of hyperoxia-induced lung injury. The presence of CO (250 ppm) prolonged the survival of rats in a hyperoxic (>95% O 2 ) environment, and inhibited the appearance of markers of hyperoxia-induced lung injury (i.e. hemorrhage, fibrin deposition, edema, airway protein accumulation, and BALF neutrophil influx) [11] . Furthermore, in a mouse model, CO inhibited the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice induced by the hyperoxia treatment. Using genedeleted mice, Otterbein and colleagues also observed that the protection afforded by CO in this model, similar to a lipopolysaccharide-induced model of lung injury, depended on the p38 MAPK pathway (Otterbein et al., unpublished observation, as reviewed in [3] ).

In direct contrast to these studies, the group of Piantadosi and colleagues reported no significant difference in the hyperoxia tolerance of rats at CO doses between 50 and 500 ppm [120] . In their model, CO did not alter the accumulation of fluid in the airway. Furthermore, CO, when applied in combination with hyperoxia, increased the activity of myeloperoxidase, a marker of airway neutrophil influx. This study also suggested that inhalation of CO (50-500 ppm) did not alter the expression of HO-1 or other antioxidant enzymes such as Manganese superoxide dismutase (MnSOD) in vivo [120] . Furthermore, Piantadosi and colleagues were able to induce oxygen tolerance in rats and HO-1 expression with hemoglobin treatment, but this tolerance also occurred in the presence of HO inhibitors, thereby not supporting a role for HO activity in oxygen tolerance [121] . Although no consensus has been reached as to the protective role of CO inhalation and/or HO-1 induction in hyperoxic lung injury, human studies will be required to show if CO will supersede NO in providing a significant therapeutic benefit in the context of severe lung diseases [122] . While antioxidant therapies have been examined, until now no human studies exist on the role of HO-1 and CO in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia [123] .

Lung transplantation is the ultimate and often last therapeutic option for several end-stage lung diseases. After lung transplantation, there remains an ongoing hazardous situation in which both acute and chronic graft failure, as well as complications of the toxic immunosuppressive regimen used (i.e. severe bacterial, fungal, and viral infections; renal failure; and Epstein-Barr-virus-related lymphomas), determine the outcome [124] . The development of chronic graft failure, obliterative bronchiolitis (OB), determines the overall outcome after lung transplantation. OB, which may develop during the first months after transplantation, is the main cause of morbidity and death following the first half-year after transplantation, despite therapeutic intervention. Once OB has developed, retransplantation remains the only therapeutic option available [124, 125] . Little is known about the pathophysiological background of OB. The possible determinants of developing OB include ongoing immunological allograft response, HLADR mismatch, cytomegalovirus infection, acute rejection episodes, organ-ischemia time, and recipient age [125] . OB patients displayed elevated neutrophil counts in the BALF, and evidence of increased oxidant activity, such as increased methionine oxidation in BALF protein and decreases in the ratio of GSH to oxidized glutathione (GSSG) in epithelial lining fluid. [126, 127] .

So far, only very limited research data are available on the possible role for HO-1 in allograft rejection after lung transplantation. Higher HO-1 expression has been detected in alveolar macrophages from lung tissue in lung transplant recipients with either acute or chronic graft failure than in stable recipients [128] . The protective role of HO-1 against allograft rejection has been shown in other transplantation models, in which solid organ transplantation typically benefits from HO-1 modulation. A higher expression of protective genes such as HO-1 has been observed in episodes of acute renal allograft rejection [129] . Furthermore, the induction of HO-1 alleviates graft-versus-host disease [130] . Adenoviral-HO-1 gene therapy resulted in remarkable protection against rejection in rat liver transplants [131] . The upregulation of HO-1 protected pancreatic islet cells from Fas-mediated apoptosis in a dose-dependent fashion, supporting an anti-apoptotic function of HO-1 [132, 133] . HO-1 may confer protection in the early phase after transplantation by inducing Th2-dependent cytokines such as IL-4 and IL-10, while suppressing interferon-γ and IL-2 production, as demonstrated in a rat liver allograft model [134] .

Beneficial effects of HO-1 modulation have also been described in xenotransplantation models, in which HO-1 gene expression appears functionally associated with xenograft survival [135] . In a mouse-to-rat heart trans-plant model, the effects of HO-1 upregulation could be mimicked by CO administration, suggesting that HOderived CO suppressed the graft rejection [136] . The authors proposed that CO suppressed graft rejection by inhibition of platelet aggregation, a process that facilitates vascular thrombosis and myocardial infarction.

HO-1 may also contribute to ischemic preconditioning, a process of acquired cellular protection against ischemia/ reperfusion injury, as observed in guinea pig transplanted lungs [137] . HO-1 overexpression provided potent protection against cold ischemia/reperfusion injury in a rat model through an anti-apoptotic pathway [138, 139] . The induction of HO-1 in rats undergoing liver transplantation with cobalt-protoporphyrin or adenoviral-HO-1 gene therapy resulted in protection against ischemia/ reperfusion injury and improved survival after transplantation, possibly by suppression of Th1-cytokine production and decreased apoptosis after reperfusion [140, 141] . Until now, no reports have addressed E-CO measurements in lung transplantation, where it is possible that differences in E-CO will be found in patients with acute and chronic allograft rejection.

The evolution of CO in exhaled breath may serve as a general marker and diagnostic indicator of inflammatory disease states of the lung, though more research will be required to verify its reliability. Increases in exhaled CO presumably reflect changes in systemic and airway heme metabolic activity from the action of HO enzymes. Evidence from numerous in vitro and animal studies indicates that HO-1 provides a protective function in many, if not all, diseases that involve inflammation and oxidative stress. Thus, the exploitation of HO-1 for therapeutic gain could be achieved through the modulation of HO-1 enzyme activity or its up-and downstream regulatory factors, either by gene transfer, pharmacological inducers, or direct application of CO by gas administration or chemical delivery [142] [143] [144] [145] . The CO-releasing molecules (transition metal carbonyls) developed by Motterlini et al. [144] show promise in the pharmacological delivery of CO for therapeutic applications in vascular and immune regulation. The CO-releasing molecules have been shown to limit hypertension in vivo and promote vasorelaxation in isolated heart and aortic rings [144] .

Ultimately, the challenge remains in applying the therapeutic potentials of HO-1 to the treatment of human diseases. In vivo models of transplantation have shown that HO-1 gene therapy protects against allograft rejection [129, 134] . Given the toxic therapy that every transplant patient receives, especially after lung transplantation, the field of transplantation medicine may bring the first frontier for human applications of HO-1 gene therapy or exogenous CO administration. The potential use of inhalation CO as a clinical therapeutic in inflammatory lung diseases has also appeared on the horizon. In one promising study, an inhalation dose of 1500 ppm CO at the rate of 20 times per day for a week produced no cardiovascular side effects [146] . Cigarette smoking and CO inhalation at identical intervals produced comparable Hb-CO levels of approximately 5%. The question of whether or not CO can be used as an inhalation therapy will soon be replaced by questions of "how much, how long, and how often?" The fear of administering CO must be weighed against the severe toxicity of the immunosuppressive agents in current use, and the often negative outcome of solid organ transplantation. Technical Description of RODS: A Real-time Public Health Surveillance System This report describes the design and implementation of the Real-time Outbreak and Disease Surveillance (RODS) system, a computer-based public health surveillance system for early detection of disease outbreaks. Hospitals send RODS data from clinical encounters over virtual private networks and leased lines using the Health Level 7 (HL7) message protocol. The data are sent in real time. RODS automatically classifies the registration chief complaint from the visit into one of seven syndrome categories using Bayesian classifiers. It stores the data in a relational database, aggregates the data for analysis using data warehousing techniques, applies univariate and multivariate statistical detection algorithms to the data, and alerts users of when the algorithms identify anomalous patterns in the syndrome counts. RODS also has a Web-based user interface that supports temporal and spatial analyses. RODS processes sales of over-the-counter health care products in a similar manner but receives such data in batch mode on a daily basis. RODS was used during the 2002 Winter Olympics and currently operates in two states—Pennsylvania and Utah. It has been and continues to be a resource for implementing, evaluating, and applying new methods of public health surveillance. Unfortunately, conventional public health disease surveillance-which relies on physician and laboratory reporting and manual analysis of surveillance data-is ill equipped for timely detection of such threats. 3 The reportable disease system relies on health care professionals to recognize, diagnose, and report cases and suspected outbreaks to public health officials 4, 5 ; however, it is unlikely that without an event or alert to raise his or her index of suspicion, a physician will attribute the early symptoms and signs of disease in a bioattack victim appropriately and report the case. 6 A key limitation of the current system is that the lone physician is blind to the cases his or her colleagues in a nearby hospital are seeing-knowledge that might lead the physician to consider uncommon diseases more strongly in his or her diagnostic reasoning. Mandatory laboratory reporting 4 is also illequipped for early detection, because it takes time before tests are ordered and specimens are obtained, transported, processed, and resulted.

Sufficiently early detection of a biological attack may be accomplished through surveillance schemes that can detect infected individuals earlier in the disease process. For completeness, we note that biosensors are being developed (and deployed) that detect organisms in the air and that this type of detection, if feasible, occurs fundamentally much earlier, because the delay introduced by the incubation period of the disease is eliminated from the surveillance system. 7 However, such approaches face unsolved technical problems in the analysis of contaminated specimens (the norm in air sampling). Biosensors also need to be in the right place-on every person's lapel or every street corner and hallway-to provide complete surveillance coverage.

Surveillance methods that can detect disease at an earlier stage are an important research direction for public health surveillance. These methods are generally referred to as syndromic surveillance because they have the goal of recognition of outbreaks based on the symptoms and signs of infection and even its effects on human behavior prior to first contact with the health care system. 8 Because the data used by syndromic surveillance systems cannot be used to establish a specific diagnosis in any particular individual, syndromic surveillance systems must be designed to detect signature patterns of disease in a population to achieve sufficient specificity. For example, it would be absurd to use only the symptom of fever to attempt to establish a working diagnosis of inhalational anthrax in an individual, but it would be very reasonable to establish a working diagnosis of anthrax release in a community if we were to observe a pattern of 1,000 individuals with fever distributed in a linear streak across an urban region consistent with the prevailing wind direction two days earlier. It would be beyond reasonable and, in fact, imperative to establish a working diagnosis of public health emergency if presented with such information.

One recent example of a form of syndromic surveillance is drop-in surveillance-the stationing of public health workers in emergency departments (EDs) and special clinics during high-profile events such as the Super Bowl to capture data on patients presenting with symptoms potentially indicative of bioterrorism. The major disadvantage of this approach is the cost of round-the-clock staffing for manual data collection.

A less expensive approach-and the one taken in the Realtime Outbreak and Disease Surveillance (RODS) system-is detection based on data collected routinely for other purposes. Examples of such data include absenteeism data, sales of over-the-counter (OTC) health care products, and chief complaints from EDs. 9 The expenses of manual data collection are avoided; however, the data obtained typically are noisy approximations of what could be obtained by direct interviewing of the patient (in the case of individual level data). Both approaches may play complementary roles with current methods of public health surveillance 10-12 by assisting the physician and public health official with a continuously updated picture of the ''health status'' of a population. 13, 14 A focus of our research has been syndromic surveillance from free-text chief complaints routinely collected by triage nurses in EDs and acute care clinics during patient registration. We have deployed this type of surveillance at the 2002 Winter Olympics and in the States of Pennsylvania and Utah. We described a previous version of the RODS system, 12 but the system has undergone considerable subsequent development both architecturally and functionally. This report provides a detailed description of the current version of RODS, an example of a computer-based public health surveillance system that adheres to the National Electronic Disease Surveillance System (NEDSS) specifications of the Centers for Disease Control and Prevention (CDC). 15, 16 Background

The role of public health surveillance is to collect, analyze, and interpret data about biological agents, diseases, risk factors, and other health events and to provide timely dissemination of collected information to decision makers. 17 Conventionally, public health surveillance relies on manual operations and off-line analysis.

Existing syndromic surveillance systems include the CDC's drop-in surveillance systems, 8 Early Notification of Community-based Epidemics (ESSENCE), 10,18 the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS), 19 the Rapid Syndrome Validation Project (RSVP), 20 and the eight systems discussed by Lober et al. 11 Lober et al. summarized desirable characteristics of syndromic surveillance systems and analyzed the extent to which systems that were in existence in 2001 had those characteristics. 11 A limitation of most systems (e.g., ESSENCE, 10 Children's Hospital in Boston, 11 University of Washington 11 ) was batch transfer of data, which may delay detection by as long as the time interval (periodicity) between batch transfers. For example, a surveillance system with daily batch transfer may delay by one day the detection of an outbreak.

Some systems required manual data input (e.g., CDC's dropin surveillance systems, RSVP, 20 and LEADERS 19 ), which is labor-intensive and, in the worst case, requires round-theclock staffing. Manual data input is not a feasible mid-or long-term solution even if the approach is to add items to existing encounter forms (where the items still may be ignored by busy clinicians).

A third limitation for existing surveillance systems is that the systems may not exploit existing standards or communication protocols like Heath Level 7 (HL7) even when they are available.

The data type most commonly used among surveillance systems is symptoms or diagnoses of patients from ED and/or physician office visits. Other types of data identified in that study include emergency call center and nurse advice lines. Other types of data being used include sales of over-thecounter health care products, prescriptions, telephone call volumes to health care providers and drug stores, and absenteeism. We have conducted studies demonstrating that the free-text chief complaint data that we use correlate with outbreaks. 21, 22 Design Objectives

The overall design objective for RODS is similar to that of an early warning system for missile defense; namely, to collect whatever data are required to achieve early detection from as wide an area as necessary and to analyze the data in a way that they can be used effectively by decision makers. It is required that this analysis be done in close to real time. This design objective is complex and difficult to operationalize because of the large number of organisms and the even larger number of possible routes of dissemination all requiring potentially different types of data for their detection, different algorithms, and different time urgencies. For this reason, our focus since beginning the project in 1999 has been on the specific problem of detecting a large-scale outbreak due to an outdoor (outside buildings) aerosol release of anthrax. Additional design objectives were adherence to NEDSS standards to ensure future interoperability with other types of public health surveillance systems, scalability, and that the system could not rely on manual data entry, except when it was done in a focused way in response to the system's own analysis of passively collected data.

This report describes RODS 1.5, which was completely rewritten as a Java 2 Enterprise Edition (J2EE) application since the previous publication describing it. RODS 1.5 is multidata type enabled, which means that any time series data can be incorporated into the databases and user interfaces. The deployed RODS system currently displays and analyzes health care delivery site registrations and separately monitors sales of OTC health care products.

Overview RODS uses clinical data that are already being collected by health care providers and systems during the registration process. When a patient arrives at an ED (or an InstaCare in Utah), the registration clerk or triage nurse elicits the patient's reason for visit (i.e., the chief complaint), age, gender, home zip code, and other data and enter the data in a registration computer. The registration computer then generates an HL7 ADT (admission, discharge, and transfer) message and transmits it to the health system's HL7 message router (also called an integration engine). There usually is only one message router per health system even if there are many hospitals and facilities. These processes are all routine existing business activities and do not need to be created de novo for public health surveillance. Figure 1 shows the flow of clinical data to and within RODS. The hospital's HL7 message router, upon receipt of an HL7 message from a registration computer, deletes identifiable information from the message and then transmits it to RODS over a secure virtual private network (VPN), or a leased line, or both (during the 2002 Winter Olympics we utilized both types of connections to each facility for fault tolerance). The RODS HL7 listener maintains the connection with the health system's message router and parses the HL7 message as described in more detail below. It then passes the chief complaint portion of the message to a Bayesian text classifier that assigns each free-text chief complaint to one of seven syndromic categories (or to an eighth category, other). The database stores the category data, which then are used by applications such as detection algorithms and user interfaces.

Data about sales of OTC health care products are processed separately by the National Retail Data Monitor, which is discussed in detail in another article in this issue of JAMIA. 23 The processing was kept separate intentionally because, in the future, the servers for the National Retail Data Monitor may operate in different physical locations than RODS. The RODS user interfaces can and do display sales of OTC health care products as will be discussed, but other user interfaces can be connected to the National Retail Data Monitor as well.

Prior to September 2001, RODS received data only from hospitals associated with the UPMC Health System, and efforts to recruit other hospitals met with resistance. After the terrorist attacks (including anthrax) in the Fall of 2001, other hospitals agreed to participate. Although data in this project are de-identified, certain information such as the number of ED visits by zip code were considered proprietary information by some health systems. Health Insurance Portability and Accountability Act (HIPAA) concerns also were very prominent in the discussions. Data-sharing agreements were executed with every participating health system that addressed these concerns. As an additional precaution, all RODS project members meet annually with University of Pittsburgh council to review obligations and are required to sign an agreement every year stating that they understand the terms of the data-sharing agreements and agree to abide by the terms. RODS began as a research project at the University of Pittsburgh in 1999 and has functioned with IRB approvals since that time.

Health care facilities send admission, discharge, and transfer (ADT) HL7 messages to RODS for patient visits in EDs and walk-in clinics. A minimal data set is sent, as shown in Figure 2 , which qualifies as a HIPAA Limited Data Set. 24 Currently the data elements are age (without date of birth), gender, home zip code, and free-text chief complaint.

The HL7 listener receives HL7 messages from the message routers located in each health system. The HL7 listener then passes the received HL7 message to the HL7 parser bean, an Enterprise JavaBean (EJB) in the RODS business logic tier. The HL7 parser bean uses regular expressions to parse the fields in an HL7 message. The HL7 parser bean then stores the parsed elements into a database through a managed database connection pool.

Although nearly all health systems utilize the HL7 messaging standard, the location of individual data elements in an HL7 message may differ from health system to health system. For example, some care providers' systems record free-text chief complaint in the DG1 segment instead of the PV2 segment of an HL7 message. To resolve this mapping problem, a configuration file written in eXtensible Markup Language (XML), a standard protocol often used to define hierarchical data elements, defines where each of the data elements can be found in the HL7 message. When an HL7 listener starts up, it reads the hospital-dependent configuration file and passes the configuration information to the parser bean.

We also use this configuration file to define the database table and field in which the HL7 parser bean should store each data element. This approach is useful because it allows the HL7 data to be stored to an external database. We anticipate that health departments with existing NEDSS or other public health surveillance databases may wish to use just this component of RODS for real-time collection of clinical data.

For hospitals that do not have HL7 message routers (two of approximately 60 in our experience to date), RODS accepts ED registration data files through either a secure Web-based data upload interface or a secure file transfer protocol. In general, these types of data transfers are technically trivial and for that reason are used by many groups but do not have the reliability of a HL7 connection (and have very undesirable time latencies).

RODS checks the integrity of the data in the HL7 messages that it receives. This processing is necessary because hospital data flows may have undesirable characteristics such as duplicates. RODS identifies and deletes duplicates by using a database trigger that creates a composite primary key before inserting the data. RODS also filters out scheduling messages, which are identified by the fact that they have future admitted date and time.

RODS monitors all data feeds to ensure continuous connections with health systems. If RODS does not receive data for six hours, it sends an alert to the RODS administrator and the sending health system's administrator. Because the commercial message routers that hospitals use queue up HL7 messages when encountering networking or system problems, data integrity is preserved.

RODS uses an Oracle8i database to store ED registration data.

(Oracle, Redwood Shores, CA). To ensure fast response for an online query (e.g., the daily counts of respiratory syndrome in a county for the past six months), we developed a cache

For connectivity with the HL7 message routers, we utilize hardware-based routers. The VPN router is a Cisco PIX 501 and the leased-line routers are a pair of Cisco 2600s (Cisco Systems, Inc., San Jose, CA).

All of the RODS processes can be run on a single computer, but in our current implementation-serving Pennsylvania F i g u r e 2. Sample HL7 admission, discharge, and transfer (ADT) message from an emergency department. The circled fields are age, gender, home zip code, admitted date and time, and free-text chief complaint, respectively.

and Utah as an application service provider-we use five dedicated servers: firewall, database, Web server, a geographic information system (GIS) server, and computation. The processes are written in Java code and can run on most platforms, but here we describe the specific platforms we use to indicate approximate sizing and processing requirements.

We developed RODS applications using the Java 2 Enterprise Edition Software Toolkit (J2EE SDK) from Sun Microsystems for cross-platform Java application development and deployment. 26 We followed contemporary application programming practices-a multitiered application consisting of a client tier (custom applications such as HL7 listeners and detection algorithms), business logic tier, database tier, and Web tier.

Business logic such as the HL7 parser bean was implemented as Enterprise JavaBeans (EJBs). NEDSS specifies EJB as the standard for application logic. RODS uses Jboss, an opensource J2EE application server, to run all EJBs. 10 The Web tier comprises the graphical user-interface to RODS and uses Java Server Pages (JSP), Java Servlets, and ArcIMS. The database tier was implemented in Oracle 8i.

RODS uses a naive Bayesian classifier called Complaint Coder (CoCo) to classify free-text chief complaints into one of the following syndromic categories: constitutional, respiratory, gastrointestinal, neurological, botulinic, rash, hemorrhagic, and other. CoCo computes the probability of each category, conditioned on each word in a free-text chief complaint and assigns a patient to the category with the highest probability. 27 The probability distributions used by CoCo are learned from a manually created training set. CoCo can be retrained with local data, and it can be trained to detect a different set of syndromes than we currently use. CoCo runs as a local process on the RODS database server. CoCo was developed at the University of Pittsburgh and is available for free download at <http://health.pitt.edu/rods/sw>.

Over the course of the project, RODS has used two detection algorithms. These algorithms have not been formally field tested because the emphasis of the project to date has been on developing the data collection infrastructure more than field testing of algorithms.