Simon T. Abrams

Circulating Histones Are Mediators of Trauma-associated Lung Injury

RATIONALE Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. OBJECTIVES To investigate the pathological roles of circulating histones in trauma-induced lung injury. METHODS Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models. MEASUREMENTS AND MAIN RESULTS In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. CONCLUSIONS This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

Human CRP Defends against the Toxicity of Circulating Histones

C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP–histone complexes in serum from patients with both elevated CRP and histones. CRP is able to compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans.

Circulating Histones Are Major Mediators of Cardiac Injury in Patients With Sepsis.

Objective:To investigate the impact of circulating histones on cardiac injury and dysfunction in a murine model and patients with sepsis. Design:Prospective, observational clinical study with in vivo and ex vivo translational laboratory investigations. Setting:General ICU and university research laboratory. Subjects:Sixty-five septic patients and 27 healthy volunteers. Twelve-week-old male C57BL/6N mice. Interventions:Serial blood samples from 65 patients with sepsis were analyzed, and left ventricular function was assessed by echocardiography. Patients’ sera were incubated with cultured cardiomyocytes in the presence or absence of antihistone antibody, and cellular viability was assessed. Murine sepsis was initiated by intraperitoneal Escherichia coli injection (108 colony-forming unit/mouse) in 12-week-old male C57BL/6N mice, and the effect of antihistone antibody (10 mg/kg) was studied. Murine blood samples were collected serially, and left ventricular function was assessed by intraventricular catheters and electrocardiography. Measurements and Main Results:Circulating histones and cardiac troponins in human and murine plasma were quantified. In 65 patients with sepsis, circulating histones were significantly elevated compared with healthy controls (n = 27) and linearly correlated with cardiac troponin T levels (rs = 0.650; p < 0.001), noradrenaline doses required to achieve hemodynamic stability (rs = 0.608; p < 0.001), Sequential Organ Failure Assessment scores (p = 0.028), and mortality (p = 0.008). In a subset of 36 septic patients without prior cardiac disease, high histone levels were significantly associated with new-onset left ventricular dysfunction (p = 0.001) and arrhythmias (p = 0.01). Left ventricular dysfunction only predicted adverse outcomes when combined with elevated histones or cardiac troponin levels. Furthermore, patients’ sera directly induced histone-specific cardiomyocyte death ex vivo, which was abrogated by antihistone antibodies. In vivo studies on septic mice confirmed the cause-effect relationship between circulating histones and the development of cardiac injury, arrhythmias, and left ventricular dysfunction. Conclusion:Circulating histones are novel and important mediators of septic cardiomyopathy, which can potentially be utilized for prognostic and therapeutic purposes.

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.

Circulating Histone Concentrations Differentially Affect the Predominance of Left or Right Ventricular Dysfunction in Critical Illness

Objectives:Cardiac complications are common in critical illness and associated with grave consequences. In this setting, elevated circulating histone levels have been linked to cardiac injury and dysfunction in experimental models and patients with sepsis. The mechanisms underlying histone-induced cardiotoxicity and the functional consequences on left ventricle and right ventricle remain unclear. This study aims to examine dose-dependent effects of circulating histones on left ventricle and right ventricle function at clinically relevant concentrations. Design:Prospective laboratory study with in vitro and in vivo investigations. Setting:University research laboratory. Subjects:Twelve-week old male C57BL/6N mice. Interventions:Cultured cardiomyocytes were incubated with clinically relevant histone concentrations, and a histone infusion mouse model was also used with hemodynamic changes characterized by echocardiography and left ventricle/right ventricle catheter–derived variables. Circulating histones and cardiac troponin levels were obtained from serial blood samples. Measurements and Main Results:IV histone infusion caused time-dependent cardiac troponin elevation to indicate cardiac injury. At moderate sublethal histone doses (30 mg/kg), left ventricular contractile dysfunction was the prominent abnormality with reduced ejection fraction and prolonged relaxation time. At high doses (≥ 60 mg/kg), pulmonary vascular obstruction induced right ventricular pressure increase and dilatation, but left ventricular end-diastolic volume improved because of reduced blood return from the lungs. Mechanistically, histones induced profound calcium influx and overload in cultured cardiomyocytes with dose-dependent detrimental effects on intracellular calcium transient amplitude, contractility, and rhythm, suggesting that histones directly affect cardiomyocyte function adversely. However, increasing histone-induced neutrophil congestion, neutrophil extracellular trap formation, and thrombosis in the pulmonary microvasculature culminated in right ventricular dysfunction. Antihistone antibody treatment abrogated histone cardiotoxicity. Conclusions:Circulating histones significantly compromise left ventricular and right ventricular function through different mechanisms that are dependent on histone concentrations. This provides a translational basis to explain and target the spectral manifestations of cardiac dysfunction in critical illness.

Histone-Associated Thrombocytopenia in Patients Who Are Critically Ill

Thrombocytopenia is observed in approximately 30% to 40% of intensive care unit (ICU) patients and associated with poor outcomes. Histones induce profound thrombocytopenia in mice,1,2 and are associated with organ injury when released following extensive cell damage in critically ill patients. 3,4 We explored the association between circulating histones and thrombocytopenia in ICU patients.

Current Pathological and Laboratory Considerations in the Diagnosis of Disseminated Intravascular Coagulation

Systemically sustained thrombin generation in vivo is the hallmark of disseminated intravascular coagulation (DIC). Typically, this is in response to a progressing disease state that is associated with significant cellular injury. The etiology could be infectious or noninfectious, with the main pathophysiological mechanisms involving cross-activation among coagulation, innate immunity, and inflammatory responses. This leads to consumption of both pro- and anticoagulant factors as well as endothelial dysfunction and disrupted homeostasis at the blood vessel wall interface. In addition to the release of tissue plasminogen activator (tPA) and soluble thrombomodulin (sTM) following cellular activation and damage, respectively, there is the release of damage-associated molecular patterns (DAMPs) such as extracellular histones and cell-free DNA. Extracellular histones are increasingly recognized as significantly pathogenic in critical illnesses through direct cell toxicity, the promotion of thrombin generation, and the induction of neutrophil extracellular trap (NET) formation. Clinically, high circulating levels of histones and histone-DNA complexes are associated with multiorgan failure, DIC, and adverse patient outcomes. Their measurements as well as that of other DAMPs and molecular markers of thrombin generation are not yet applicable in the routine diagnostic laboratory. To provide a practical diagnostic tool for acute DIC, a composite scoring system using rapidly available coagulation tests is recommended by the International Society on Thrombosis and Haemostasis. Its usefulness and limitations are discussed alongside the advances and unanswered questions in DIC pathogenesis.

Accuracy of circulating histones in predicting persistent organ failure and mortality in patients with acute pancreatitis

Early prediction of acute pancreatitis severity remains a challenge. Circulating levels of histones are raised early in mouse models and correlate with disease severity. It was hypothesized that circulating histones predict persistent organ failure in patients with acute pancreatitis.

The clinical utility of fibrin-related biomarkers in sepsis.

Sepsis is associated with systemic inflammatory responses and induction of intravascular fibrin formation. Our aim is to investigate whether three fibrin-related markers (FRM) reflect the extent of coagulation activation in vivo and evaluate their clinical usefulness in identifying as well as monitoring patients with sepsis. Fibrin-degradation products (FDP), D-dimer and soluble fibrin monomer assays were measured on plasma samples from patients in the ICU with sepsis (n = 37), systemic inflammatory response syndrome (SIRS) (n = 35) and healthy individuals (n = 15). The levels were correlated with each other and also with fibrinogen, prothrombin time, platelets and antithrombin III. Clinical correlation was also performed for the diagnosis of sepsis and longitudinal monitoring for survival or death.There was strong correlation between the three FRM (r = 0.38–0.93, P < 0.0001) with only fibrin monomer correlating significantly with prothrombin time, fibrinogen and platelet levels. Clinically, all three FRM could discriminate between patients with sepsis, SIRS and healthy individuals with FDP, and D-dimer showing statistical significance (P < 0.05). No FRM predicted outcome from a single measurement but FDP was significantly able to predict patient survival from serial samples [mean FDP (&mgr;g/ml) from 35.36 to 21.37 (first to third ICU-day), P < 0.05]. Fibrin monomer appears the most sensitive indicator of coagulation activation, whereas D-dimer and FDP levels can significantly differentiate ICU patients with sepsis from those without. In addition, FDP would be preferable for monitoring with its statistically significant time-dependent prediction of survival or death from sepsis.

Lipoproteins, statins and septic haematology patients

The recent review by Cohen and Drage (2011) provides a very thorough and practical approach to the management of sepsis in haematology patients. However, two points were possibly overlooked in this comprehensive article – (i) the important role of biphasic activated partial thromboplastin waveform (BPW) as an early predictor of sepsis, and (ii) the increasingly recognized, beneficial effects of the cholesterol-lowering agents, statins, in septic and critically ill patients. BPW was demonstrated initially by our group (Toh et al, 2003) and later confirmed by several other investigators to be an early predictor of sepsis. More importantly, this abnormal laboratory phenomenon has been shown to correlate with the development of disseminated intravascular coagulation (DIC), probably the worst complication of sepsis (Downey et al, 1998). BPW is an abnormality of the optical transmission waveform obtained during measurement of the activated partial thromboplastin time as part of the routine coagulation screen and, as such, is an inexpensive, rapid, and repeatable tool. BPW is also beneficial with respect to the clinical deterioration of a haemato-oncology patient, to decide whether the worsening condition is related to infection or systemic inflammatory response secondary to other causes, such as chemotherapy-related adverse events. In an 8-month study of 187 consecutive intensive care patients (not specific to haematology) with analyses of BPW, procalcitonin and C-reactive protein (CRP), BPW on day one was significantly more abnormal in patients with severe sepsis or septic shock than in patients with systemic inflammatory response syndrome (Chopin et al, 2006). Two other clinical conundrums in patients with neutropenic fever are the development of clinical signs of sepsis without microbiology proven evidence of infection; and negative blood culture results in the presence of sepsis. Hussain et al (2008) analysed 49 consecutive neutropenic haematological patients over a 10month period and identified the biphasic transmittance waveform (bTW; derivation of BPW) as a rapid marker of sepsis. Interestingly, the bTW results preceded the blood culture results by up to three days, a crucial time in patients with severe sepsis. Thus, BPW can be helpful in deciding the need for antimicrobial therapy in these patients and avoid their unnecessary prescription, reducing the risks of cytopenias, renal impairment and clostridium difficile-associated diarrhoea. The review elaborates on procalcitonin as a marker of severe sepsis. However in addition to lack of availability in many hospitals, and high cost, procalcitonin has not fared as well as BPW in predicting severe sepsis. In the study by Chopin et al (2006), among the comparison between C-reactive protein (CRP), procalcitonin and BPW, the latter was more accurate for differentiating patients with severe sepsis and septic shock, with 90% sensitivity and 92% negative predictive value. A more recent paper commented on the combination of BPW and procalcitonin to fare even better, with sensitivity of 79%, specificity of 96% and negative predictive value of 96% for the diagnosis of sepsis (Zakariah et al, 2008). The molecular basis responsible for the development of BPW has been shown by our group to be due to calcium-dependent formation of a complex of CRP and very low-density lipoprotein (VLDL). Given that BPW correlates with poor outcome in septic patients, drugs that affect lipids and CRP levels (e.g. statins) may modify the CRP-VLDL complex formation and thus the sepsis phenomenon, proving to be a cost-effective therapeutic strategy in these situations. Statins have been increasingly identified to have more than just lipid-lowering properties, some of which have beneficial effects in septic and critically ill patients. The two crucial properties in this regard are their anti-inflammatory potential and effects on the coagulation system. Inflammation-coagulation crosstalk is a key component determining the development of DIC and thus, the outcome of septic patients. As such, modifying both these factors can be beneficial in the clinical course of septic patients, as evidenced by the systematic review and meta-analysis of statins for the prevention and treatment of infections. (Tleyjeh et al, 2009). It should however be stressed that further large-scale trials are required before statins can be accepted for anti-infective purposes, possibly in conjunction with existing antimicrobial therapies and should not be considered replacements for these agents in critically-ill haematology subjects. The beneficial effect of statins in critically ill haematology patients may go beyond just the ‘anti-infective’ properties. These agents are increasingly recognized to be relevant in preventing the development of venous thromboembolism (VTE; Glynn et al, 2009). VTE is a prominent cause of morbidity and mortality in critically ill patients and strict thromboprophylaxis protocols are in place in most units. However, haematology patients present additional challenges in that many of these patients are thrombocytopenic, making pharmacological anticoagulation difficult, and have additional risk factors of malignancy and chemotherapeutic agents, which can lead to anticoagulation failure. In these circumstances, statins can be favourable with their absent myelosuppressive effects and minimal drug interactions. correspondence