Mikhail A. Sovershaev

Infusion of methylene blue in human septic shock: A pilot, randomized, controlled study

ObjectiveTo evaluate the effects of continuous infusion of methylene blue (MB), an inhibitor of the nitric oxide pathway, on hemodynamics and organ functions in human septic shock. DesignProspective, randomized, controlled, open-label, pilot study. SettingMultidisciplinary intensive care unit of a university hospital. PatientsTwenty patients with septic shock diagnosed <24 hrs before randomization. InterventionsPatients were randomized 1:1 to receive either MB (MB group, n = 10) or isotonic saline (control group, n = 10), adjunctive to conventional treatment. MB was administered as an intravenous bolus injection (2 mg/kg), followed 2 hrs later by infusion at stepwise increasing rates of 0.25, 0.5, 1, and 2 mg/kg/hr that were maintained for 1 hr each. During infusion, mean arterial pressure was maintained between 70 and 90 mm Hg, while attempting to reduce concurrent adrenergic support. Measurements and Main Results Hemodynamics and organ function variables were assessed over a 24-hr period, and the survival rate at day 28 was noted. Infusion of MB prevented the stroke volume and the left-ventricular stroke work indexes from falling and increased mean arterial pressure. Compared with the control group, MB reduced the requirement for norepinephrine, epinephrine, and dopamine by as much as 87%, 81%, and 40%, respectively. Oxygen delivery remained unchanged in the MB group and decreased in the control group. MB also reduced the body temperature and the plasma concentration of nitrates/nitrites. Leukocytes and organ function variables such as bilirubin, alanine aminotransferase, urea, and creatinine were not significantly affected. Platelet count decreased in both groups. Five patients treated with MB survived vs. three patients receiving conventional treatment. ConclusionsIn human septic shock, continuously infused MB counteracts myocardial depression, maintains oxygen transport, and reduces concurrent adrenergic support. Infusion of MB appears to have no significant adverse effects on the selected organ function variables.

Extravascular lung water determined with single transpulmonary thermodilution correlates with the severity of sepsis-induced acute lung injury

Objective:To find out if the extravascular lung water index (EVLWI) and the derived permeability indexes determined by the single transpulmonary thermodilution technique are associated with markers of acute lung injury in human septic shock. Design:Prospective, observational study. Setting:Mixed intensive care unit of a 900-bed university hospital. Patients:Thirty-eight consecutive adult patients with septic shock and acute lung injury. Interventions:None. Measurements and Main Results:The variables were assessed over a 72-hr period and included hemodynamics, EVLWI, and pulmonary vascular permeability indexes determined with the single indicator transpulmonary thermodilution technique, lung compliance, oxygenation ratio (Pao2/Fio2), lung injury score, cell counts, and the plasma concentration of endothelin-1. At day 1, EVLWI was elevated (≥7 mL/kg) in 28 (74%) patients and correlated with lung compliance (r = −.48, p = .002), Pao2/Fio2 (r = −.50, p = .001), lung injury score (r = .46, p = .004), roentgenogram quadrants (r = .39, p = .02), and platelet count (r = −.43, p = .007). At day 3, EVLWI correlated with compliance (r = −.51, p = .002), Pao2/Fio2 (r = −.49, p = .006), and lung injury score (r = .53, p = .003). At day 3, EVLWI and pulmonary vascular permeability indexes were higher in nonsurvivors (p< .05). The plasma concentration of endothelin-1 (mean ± sd) was significantly higher in patients with elevated EVLWI (≥7 mL/kg) (3.85 ± 1.40 vs. 2.07 ± 0.38 pg/mL, respectively). Twenty-two (59%) patients died before day 28. Conclusions:In human septic shock, EVLWI demonstrated moderate correlation with markers of acute lung injury, such as lung compliance, oxygenation ratio, roentgenogram quadrants, and lung injury score. In nonsurvivors, EVLWI and permeability indexes were significantly increased at day 3. Thus, EVLWI might be of value as an indicator of prognosis and severity of sepsis-induced acute lung injury.

Intracellular and Surface Distribution of Monocyte Tissue Factor: Application to Intersubject Variability

Objective—The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). Methods and Results—After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0±3.5%) versus LRs (11.2±1.2%; P≤0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1±0.5 arbitrary units [AU]) compared with LRs (5.4±0.4 AU; P≤0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. Conclusions—The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Endothelial dysfunction enhances vasoconstriction due to scavenging of nitric oxide by a hemoglobin-based oxygen carrier

Background:To date, there is no safe and effective hemoglobin-based oxygen carrier (HBOC) to substitute for erythrocyte transfusion. It is uncertain whether a deficiency of endothelial nitric oxide bioavailability (endothelial dysfunction) prevents or augments HBOC-induced vasoconstriction. Methods:Hemodynamic effects of infusion of PolyHeme (1.08 g hemoglobin/kg; Northfield Laboratories, Evanston, IL) or murine tetrameric hemoglobin (0.48 g hemoglobin/kg) were determined in awake healthy lambs, awake mice, and anesthetized mice. In vitro, a cumulative dose-tension response was obtained by sequential addition of PolyHeme or tetrameric hemoglobin to phenylephrine-precontracted murine aortic rings. Results:Infusion of PolyHeme did not cause systemic hypertension in awake lambs but produced acute systemic and pulmonary vasoconstriction. Infusion of PolyHeme did not cause systemic hypertension in healthy wild-type mice but induced severe systemic vasoconstriction in mice with endothelial dysfunction (either db/db mice or high-fat fed wild-type mice for 4–6 weeks). The db/db mice were more sensitive to systemic vasoconstriction than wild-type mice after the infusion of either tetrameric hemoglobin or PolyHeme. Murine aortic ring studies confirmed that db/db mice have an impaired response to an endothelial-dependent vasodilator and an enhanced vasoconstrictor response to HBOC. Conclusions:Reduction in low molecular weight hemoglobin concentrations to less than 1% is insufficient to abrogate the vasoconstrictor effects of HBOC infusion in healthy awake sheep or in mice with reduced vascular nitric oxide levels associated with endothelial dysfunction. These findings suggest that testing HBOCs in animals with endothelial dysfunction can provide a more sensitive indication of their potential vasoconstrictor effects.

In-cell Western assay: a new approach to visualize tissue factor in human monocytes.

Summary.  Background: Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions. Aim: In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique – in‐cell Western (ICW) assay using an Odyssey Infrared Imaging System. Methods and results: The ICW assay of TF in resting or lipopolysaccharide (LPS)‐stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS‐stimulated MNCs as 0.09 ± 0.02 ng and 0.43 ± 0.15 ng 10−6 cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit. Conclusions: A novel TF ICW assay is a reproducible, time‐ and cost‐saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.

Selecting and deselecting imatinib-resistant clones: observations made by longitudinal, quantitative monitoring of mutated BCR-ABL

Resistance to imatinib during the treatment of chronic myeloid leukaemia (CML) is frequently associated with point mutations in the ABL gene encoding the ATP binding region likely to cause disease relapse. Early diagnosis and monitoring of these mutations may be important in order to prevent rapid expansion of resistant clones. We describe a quantitative mutation-specific PCR assay based on the readily available Taqman platform. Selectivity for the mutated target is conferred by mutation-specific primers destabilised by additional mismatches. The assay can be carried out in parallel to standard BCR-ABL quantification and is therefore more quickly compared to standard sequencing procedures. The sensitivity of the assay reaches 0.1%. It also allows for quantitative assessment of mutated clones. By analysing sequential samples of resistant subjects, we show how mutated clones were selected, maintained or deselected depending on the individual treatment setting. The high sensitivity and practical merits of this method makes it a good candidate for prospective molecular surveillance of patients at high risk for imatinib resistance.

Novel endothelin receptor antagonist attenuates endotoxin-induced lung injury in sheep.

ObjectiveTo evaluate the cardiopulmonary effects of the novel endothelin receptor antagonist tezosentan in endotoxin-induced lung injury in sheep and to assess the dose response to tezosentan and endothelin-1 in healthy sheep. DesignProspective, randomized, controlled experimental study. SettingUniversity animal laboratory. SubjectsTwenty-one yearling sheep. InterventionsSeventeen awake, chronically instrumented sheep were subjected to intravenous infusion of Ringer’s lactate for 24 hrs. The animals were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide group (n = 7) receiving an intravenous infusion of Escherichia coli lipopolysaccharide 15 ng·kg−1·min−1, and a tezosentan group (n = 7) subjected to lipopolysaccharide and, from 4 hrs, an intravenous injection of tezosentan 3 mg/kg followed by infusion of 1 mg·kg−1·hr−1. In addition, four healthy sheep, exposed to an intravenous infusion of endothelin-1 at 20 ng·kg−1·min−1, after 1 hr received tezosentan in stepwise increasing doses of 0.5, 1, and 2 mg·kg−1·hr−1 that were maintained for 1 hr each. After a 4-hr recovery, the sheep received infusions of tezosentan at the same dose rates as a pretreatment to endothelin-1. Measurements and Main ResultsIn the sham-operated sheep, all cardiopulmonary variables remained unchanged. Lipopolysaccharide caused pulmonary hypertension, increased extravascular lung water index, and induced arterial hypoxemia. Tezosentan decreased the increments in pulmonary vascular resistance and extravascular lung water index by as much as 60% and 70%, respectively. In parallel, tezosentan ameliorated arterial hypoxemia, increased cardiac index, attenuated the decrease in stroke volume index, and reduced systemic vascular resistance. Compared with the lipopolysaccharide group, tezosentan further increased plasma concentrations of endothelin-1. In healthy animals, the administration of endothelin-1 induced systemic and pulmonary hypertension, increased extravascular lung water index, and evoked bradycardia and a decrease in cardiac index. These changes were attenuated by tezosentan infused at 1 and 2 mg·kg−1·hr−1. ConclusionsIn an ovine model of endotoxin-induced lung injury, tezosentan ameliorates pulmonary hypertension, lung edema, cardiac dysfunction, and arterial hypoxemia. Tezosentan counteracts the hemodynamic effects of endothelin-1 in a dose-dependent manner.

Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C

IntroductionStudies in vitro reveal that endothelin-1 (ET-1) activates the α isoform of protein kinase C (PKC-α) in cultures of endothelial cells, thereby deranging cellular integrity. Sepsis and endotoxemia are associated with increased plasma concentrations of ET-1 that induce acute lung injury (ALI). We recently reported that non-selective ET-1 receptor blockade attenuates ALI in sheep by reducing the endotoxin-induced increase in extravascular lung water index (EVLWI). The aim of this study was to find out whether this attenuation is associated with reduced translocation of PKC-α from the cytosolic to the membrane fraction of lung tissue homogenate.MethodsSeventeen awake, instrumented sheep were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide (LPS) group (n = 7) receiving an intravenous infusion of Escherichia coli 15 ng/kg per min for 24 hours, and a tezosentan group (n = 7) subjected to LPS and, from 4 hours, an intravenous injection of tezosentan 3 mg/kg followed by infusion at 1 mg/kg per hour for the reminder of the experiment. Pulmonary micro-occlusion pressure (Pmo), EVLWI, plasma concentrations of ET-1, tumor necrosis factor-a (TNF-a), and interleukin-8 (IL-8) were determined every 4 hours. Western blotting was used to assess PKC-α.ResultsIn non-treated sheep a positive correlation was found between the plasma concentration of ET-1 and Pmo in the late phase of endotoxemia (12 to 24 hours). A positive correlation was also noticed between Pmo and EVLWI in the LPS and the LPS plus tezosentan groups, although the latter was significantly reduced in comparison with LPS alone. In both endotoxemic groups, plasma concentrations of ET-1, TNF-α, and IL-8 increased. In the LPS group, the cytosolic fraction of PKC-α decreased by 75% whereas the membrane fraction increased by 40% in comparison with the sham-operated animals. Tezosentan completely prevented the changes in PKC-α in both the cytosolic and the membrane fractions, concomitantly causing a further increase in the plasma concentrations of ET-1, TNF-α, and IL-8.ConclusionIn endotoxemic sheep, ET-1 receptor blockade alleviates lung injury as assessed by a decrease in EVLWI paralleled by a reduction in Pmo and the prevention of activation of PKC-α.

No evidence for the presence of tissue factor in high‐purity preparations of immunologically isolated eosinophils

Summary.  Background: To date, there is no unequivocal opinion on whether human eosinophils express tissue factor (TF). Therefore, we studied the expression of TF protein and activity in resting or stimulated immunologically purified human eosinophils. Methods and results: By use of immunologic isolation, we achieved over 96% purity of eosinophil preparations, and contamination by CD14‐positive cells was below 0.3%. Flow cytometric [fluorescence‐activated cell sorting (FACS)] analysis of eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. This was active TF antigen, as confirmed by TF activity assay (19.2 ± 4.2 and 28.6 ± 3.1 mU per 106 cells, stimulated in medium or plasma, respectively). We found no detectable TF mRNA levels in resting or stimulated eosinophils by real‐time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation. Conclusions: Our data indicate that there is no evidence for TF expression in high‐purity preparations of immunologically isolated eosinophils.

Regulation of tissue factor procoagulant activity by post-translational modifications

Post-translational modification of amino acid residues is a common way to regulate localization, stability and ultimately the function of the protein. Tissue factor (TF), the major initiator of blood coagulation cascade, receives several post-translational modifications, like glycosylation, phosphorylation, palmitoylation and nitrosylation. Recent studies have demonstrated that these processes play important roles in modulating biological functions of TF. The present review highlights the mechanisms of several common protein post-translational modifications of TF with the special reference on the recent knowledge about their roles in regulation of trafficking, stability as well as procoagulant and signaling functions of TF.