Yasir Alhamdi

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.

Current consideration and management of disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is a devastating clinical condition that is characterized by the loss of normal hemostatic control in response to sustained and systemic cell injury. The inciting injury may be from infection, trauma, or malignancy, but the consequent pathophysiology is multifactorial involving intertwined feedback loops between the coagulant, immune, and inflammatory pathways. Central to this is thrombin generation, but the ubiquitous nature of its in vivo functional consequences can make it difficult to dissect away the separate but overlapping components to the clinical problem. Therefore, early recognition and resolution of the precipitating events leading to DIC remains the central tenet to clinical care. This article refreshes our conceptual understanding of DIC pathogenesis and draws in recent advances in the cycle of cell death caused by extracellular nuclear proteins. It also aims to delineate recognition of response pathways that can be predominantly procoagulant or profibrinolytic to enable a more personalized and evidence-based approach to be delivered to the patient with DIC.

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.

The role of extracellular histones in haematological disorders

Over the past decades, chromosomal alterations have been extensively investigated for their pathophysiological relevance in haematological malignancies. In particular, epigenetic modifications of intra‐nuclear histones are now known as key regulators of healthy cell cycles that have also evolved into novel therapeutic targets for certain blood cancers. Thus, for most haematologists, histones are DNA‐chained proteins that are buried deep within chromatin. However, the plot has deepened with recent revelations on the function of histones when unchained and released extracellularly upon cell death or from activated neutrophils as part of neutrophil extracellular traps (NETs). Extracellular histones and NETs are increasingly recognized for profound cytotoxicity and pro‐coagulant effects. This article highlights the importance of recognizing this new paradigm of extracellular histones as a key player in host defence through its damage‐associated molecular patterns, which could translate into novel diagnostic and therapeutic biomarkers in various haematological and critical disorders.

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.

Protective Effects of Carbon Monoxide-Releasing Molecule-2 on the Barrier Function of Intestinal Epithelial Cells

Objective To investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells. Materials and Methods After pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm2 at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM). Results CORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment. Conclusions The present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.

Extracellular Histones Inhibit Complement Activation through Interacting with Complement Component 4

Complement activation leads to membrane attack complex formation, which can lyse not only pathogens but also host cells. Histones can be released from the lysed or damaged cells and serve as a major type of damage-associated molecular pattern, but their effects on the complement system are not clear. In this study, we pulled down two major proteins from human serum using histone-conjugated beads: one was C-reactive protein and the other was C4, as identified by mass spectrometry. In surface plasmon resonance analysis, histone H3 and H4 showed stronger binding to C4 than other histones, with KD around 1 nM. The interaction did not affect C4 cleavage to C4a and C4b. Because histones bind to C4b, a component of C3 and C5 convertases, their activities were significantly inhibited in the presence of histones. Although it is not clear whether the inhibition was achieved through blocking C3 and C5 convertase assembly or just through reducing their activity, the outcome was that both classical and mannose-binding lectin pathways were dramatically inhibited. Using a high concentration of C4 protein, histone-suppressed complement activity could not be fully restored, indicating C4 is not the only target of histones in those pathways. In contrast, the alternative pathway was almost spared, but the overall complement activity activated by zymosan was inhibited by histones. Therefore, we believe that histones inhibiting complement activation is a natural feedback mechanism to prevent the excessive injury of host cells.