Jordan Kountchev

Levosimendan inhibits release of reactive oxygen species in polymorphonuclear leukocytes in vitro and in patients with acute heart failure and septic shock: a prospective observational study

IntroductionLevosimendan is an extensively investigated inodilator showing also cardioprotective and antiinflammatory effects. The aim of our study was to explore the influence of levosimendan on polymorphonuclear leucocytes (PMN), a main source of reactive oxygen species, in vitro and in patients with acute heart failure or septic myocardial depression.MethodsPMN isolated from healthy volunteers were incubated with levosimendan in vitro. After stimulation with N-formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA) respiratory burst was quantified using a fluorescent dye. Apoptosis and expression of cell adhesion molecules of PMN were measured by flow cytometry. For determination of in vivo effects patients with acute heart failure (n = 16) or septic cardiac failure (n = 9) receiving levosimendan treatment were enrolled consecutively. PMN were isolated to measure respiratory burst activity before treatment as well as one and two hours after initiation of levosimendan administration. Furthermore inflammatory, hemodynamic and renal function parameters were obtained.ResultsIn vitro, levosimendan suppressed respiratory burst activity in fMLP or PMA stimulated PMN in a dose dependent manner by 30 ± 11% (P < 0.001) at 100 ng/mL and by 27 ± 17% (P < 0.001) at 1000 ng/mL respectively. Markers of apoptosis and PMN cell adhesion molecule expression remained unaffected by levosimendan treatment.In vivo, levosimendan treatment for two hours resulted in a significant reduction of PMA stimulated oxidative burst by 45% (P < 0.01) and fMLP stimulated oxidative burst by 49% (P < 0.05) in patients with acute heart failure. In patients suffering from septic shock levosimendan treatment decreased oxidative burst activity in unstimulated, fMLP and PMA stimulated PMN by 48% (P < 0.05), 46% (P < 0.01) and 43% (P < 0.01) respectively.ConclusionsLevosimendan appears to exert distinct immunomodulatory effects by decreasing oxidative burst activity of PMN. This property might contribute to the previously described cardioprotective effects of the drug.

Mechanisms of Neutrophil Transmigration Across Renal Proximal Tubular HK-2 Cells

Background: Adhesion of intratubular leukocytes to proximal tubules in biopsies of patients with rapidly progressive glomerulonephritis and the appearance of leukocytes in the urine in interstitial nephritis suggest interactions between leukocytes and tubular epithelia in renal diseases. The aim of this study was to investigate the effect of cytokines and endotoxin on leukocyte migration through proximal tubular epithelial cells and also to determine the role of the transmembrane adhesion molecules ICAM-1 and CD47 in this process. Methods: Experiments determined transepithelial migration (TEM) of PMN (polymorphonuclear) leukocytes through monolayers of HK-2. Expression of ICAM-1 and CD47 was assessed via confocal immunofluorescence, FACS analysis and western blotting. The effect of antibodies against ICAM-1 and CD47 on TEM was examined. Furthermore measurements of cytokine release (IL- 6 and IL-8) were performed. Results: Preincubation of HK-2 cells with either TNFα or LPS resulted in stimulation of PMN migration through monolayers of HK-2 cells. There was no preferred direction of transmigration. ICAM-1 was expressed by HK-2 cells and expression was increased after 4 h stimulation with TNFα or LPS. Application of ICAM-1 antibodies inhibited TEM. CD47 was expressed in both HK-2 cells and PMN. CD47 antibodies inhibited predominantly basolateral-to-apical TEM. HK-2 cells released IL-8 and IL-6 preferably into the apical compartment. Additionally, we showed that fMLP induced transmigration through monolayers of HK-2 cells was associated with significant increased CD47 expression on PMN cell surfaces. Conclusions: Inflammatory mediators stimulate TEM of PMN through monolayers of HK-2 cells without a clearly discernible preference of direction. Mechanisms involved in TEM stimulated by cytokines or endotoxin appear to be mainly changes in surface receptor densities of HK-2 cells with ICAM-1 and CD47 playing an essential role.

Calpain, calpastatin activities and ratios during myocardial ischemia-reperfusion.

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a ‘calpain burst’ impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). As expected no detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without affect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain–calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downegulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of I/R changes, although the role of calpastatin downregulation remains to be elucidated.

Lemierre's syndrome following infectious mononucleosis

ZusammenfassungWir berichten über eine 18-jährige Patientin, die nach einem protrahierten Verlauf einer Infektion der oberen Atemwege (Pharyngitis, Sinusitis) mit dem Bild einer respiratorischen Insuffizienz bei septischem Schock auf die medizinische Intensivstation in Innsbruck aufgenommen wurde. Als Fokus entpuppte sich eine abszedierende Pneumonie mit massivem Pleuraempyem. Als verantwortlicher Erreger konnte Fusobacterium necrophorum aus dem Pleurapunktat gezüchtet werden. Neben dem Bild eines Multiorganversagens (respiratorische Insuffizienz (ALI), akutes Nierenversagen, disseminierte intravaskuläre Gerinnungsstörung) entwickelte die Patienten eine Schwellung im Bereich des Halses rechts und anschließend eine septische Arthritis des rechten Sternoclaviculargelenks. Eine genaue Anamnese und Erhebung der auswärtigen Vorbefunde erbrachte den Hinweis auf eine vorangegangene Episode einer Epstein-Barr Virus Infektion. Die zuvor gesunde Frau konnte nach einem protrahierten Verlauf nach antibiotischer Therapie und mehrfachen chirurgischen Interventionen in gutem Allgemeinzustand wieder entlassen werden. Das Lemierre-Syndrom ist eine schwer verlaufende Infektionserkrankung, charakterisiert durch Pharyngitis, Sepsis und Thrombose der Vena Jugularis Interna. Als verantwortlicher Keim wird in der Mehrzahl der Fälle Fusobacterium necrophorum gefunden. Eine vorangegangene Infektion mit EBV erleichtert das Eindringen des Keimes aus dem Oropharynx in die Blutbahn.SummaryWe report the case of an 18-year-old woman who was admitted to the medical intensive care unit in Innsbruck with severe septic shock and respiratory insufficiency following a prolonged infection of the upper airways (pharyngitis, sinusitis). Abscessing pneumonia and bilateral pleural empyema were diagnosed as focus. Cultures of pleural fluids were positive for Fusobacterium necrophorum. In addition to multiple organ dysfunction syndrome (acute lung injury, acute renal failure, disseminated intravascular coagulation), she developed tenderness in the right neck followed by septic arthritis of the right sternoclavicular joint a few days later. Further history revealed a previous period of infectious mononucleosis (EBV infection). The previously healthy patient eventually made a complete recovery after prolonged treatment in the ICU including antibiotic therapy and multiple surgical interventions and drainage. Lemierres syndrome is characterized by severe infection, with pharyngitis, sepsis and thrombosis of the internal jugular vein, and is most frequently associated with upper airway infection with Fusobacterium necrophorum, often preceded by infection with Epstein–Barr virus which enables bacteria growing in the oral cavity to invade.

Disturbances of electrolytes and blood chemistry in acute alcohol intoxication.

SummaryPrevalence of electrolyte disturbances and biochemical changes were determined in patients admitted to the emergency room of the Department of Internal Medicine in Innsbruck, Austria during a six-month period. The value of biochemical parameters for the detection of chronic alcohol abuse was also investigated. The most frequent electrolyte disturbances found were hypernatremia (41%), hyperchloremia (21%), hypermagnesemia (17%) and hypocalcemia (15%), whereas hypokalemia and hypophosphatemia were observed quite rarely (5% and 3.4%, respectively). The most frequent biochemical changes observed were consistent with signs of cellular toxicity i.e. increased liver enzymes (elevated gamma-glutamyltransferase (GGT), aspartate aminotransferase, alanine aminotransferase and lactic dehydrogenase) as well as signs of pancreatitis (elevated serum lipase and amylase) and muscle damage (elevated creatine kinase). The most frequent changes in blood counts were leucocytosis (23%), thrombocytopenia (14%), and anemia (12%). C-reactive protein showed only minimal elevation. Male sex and level of blood alcohol were detected as major risk factors for the diagnosis of chronic alcohol abuse in the patient sample investigated. When testing the value of routinely measured parameters for predicting the presence of chronic alcohol abuse, GGT and mean corpuscular volume of red blood cells (MCV) appeared to be of equal value. A combination of elevated blood alcohol with an increase in either of these markers may be interpreted as high risk for chronic alcohol abuse in this particular group of patients.ZusammenfassungÜber einen Zeitraum von sechs Monaten wurden Prävalenzraten von Elektrolytstörung sowie laborchemischen Veränderung bei Patienten bestimmt, die mit der Diagnose Alkoholvergiftung in die Notfallaufnahme der Universitätsklinik für Innere Medizin in Innsbruck in den Jahren 2000/2001 eingeliefert wurden. Weiters wurde die Wertigkeit der Routineparameter MCV und GGT für die Diagnose von chronischem Alkoholabusus evaluiert. Die am häufigsten festgestellten Elektrolytstörungen waren Hypernatriämie (41%), Hyperchlorämie (21%), Hypermagnesiämie (17%) und Hypokalzämie (15%), während Hypokaliämie (5%) sowie Hypophosphatämie (3.4%) relativ selten auftraten. Die auffälligsten biochemischen Veränderungen sind vereinbar mit Zeichen der Leberzellschädigung (erhöhte Gamma-Glutamyltransferase (GGT), Glutamin-Oxalazetat-Transaminase und Laktatdehydrogenase) sowie Pankreatitis (erhöhte Serum-Amylase und -Lipase) und Muskelzellschädigung (erhöhte Kreatinkinase), eine signifikante Rhabdomyolyse trat nur in 2 Fällen auf. Auffällige Blutbildveränderungen bestanden in Leukozytose (23%), Thrombopenie (14%) und Anämie (12%). C-reaktives Protein war dabei nur geringfügig erhöht. Mittels logistischer Regression wurden männliches Geschlecht sowie Intoxikationsgrad (Blutalkoholspiegel) als wesentliche Risikofaktoren für das Bestehen von chronischem Alkoholabusus identifiziert. Bei der Suche nach einem geeigneten Routineparameter für die Diagnosestellung von chronischem Alkoholmissbrauch bei Akutpatienten erwiesen sich GGT und mittleres korpuskuläres Erythrozytenvolumen (MCV) als gleichwertig. Die Kombination von Blutalkoholwerten ≥3‰ und erhöhtem GGT bzw. MCV kann als hoch prädiktiv für das Vorhandensein von chronischem Alkoholmissbrauch angesehen werden.

Reduction of D-dimer levels after therapeutic administration of antithrombin in acquired antithrombin deficiency of severe sepsis

IntroductionIn acute disseminated intravascular coagulation, the effect of antithrombin (AT) administration on elevated levels of D-dimer is not well established. In the present study, we report on changes in circulating levels of D-dimer in response to administration of AT in a series of patients with acquired AT deficiency due to severe sepsis.MethodsEight consecutive critically ill medical patients presenting with acute disseminated intravascular coagulation associated with severe sepsis/septic shock received a single bolus infusion of AT over 30 minutes, aiming to achieve physiological AT levels. Haemostatic parameters including D-dimer were assessed prior to, 6 and 24 h after AT administration. An average of 42 ± 9 U/kg body weight was infused.ResultsFollowing AT substitution, elevated levels of D-dimer fell whereas AT levels rose.ConclusionThese observations support the notion that AT can favourably affect fibrin degradation accompanying disseminated intravascular coagulation of severe sepsis.

Influence of antithrombin on ischemia/reperfusion injury in the isolated blood-free perfused rat heart

INTRODUCTION Antithrombin (AT) is well known as an important inhibitor of the coagulation system. An interesting new hypothesis is that antithrombin exerts specific anti-inflammatory effects by stimulating the production of prostacyclin in endothelial cells. Recent studies report beneficial influence on ischemia/reperfusion injury in several organs. These effects are independent of the coagulation system. We investigated the influence of antithrombin on ischemia/reperfusion injury and prostacyclin release in the isolated rat heart. Since the perfusion of the hearts was without blood, the used model essentially describes effects of antithrombin on endothelial cells. MATERIAL AND METHODS Experiments were performed using the temperature-controlled and pressure-constant Langendorff apparatus. The hearts of 32 male Sprague-Dawley rats were subjected to 20 min of global ischemia followed by 30 min of reperfusion. Antithrombin was administered in three different concentrations (1, 4 and 8 U/ml) 15 min prior to global ischemia. Cardiac contractility parameters and biochemical parameters were measured. RESULTS Treatment with antithrombin did not increase the release of prostacyclin significantly after ischemia. Antithrombin at a concentration of 8 U/ml led to a significant increase in creatine kinase (CK; p<0.05) and troponin I (p<0.05), whereas measurements of lactate dehydrogenase (LDH) revealed no significant differences between treated and untreated hearts. CONCLUSION Our study shows that antithrombin did not reduce ischemia/reperfusion injury in the isolated heart, and prostacyclin is not significantly released following antithrombin treatment. High concentrations of antithrombin, however, might have a negative influence on the reperfused heart. The underlying mechanism remains unclear.

Between benzodiazepine over-sedation and neurological damage

In the February issue of Critical Care, Dr McKenzie and colleagues [1] describe a new method to differentiate between midazolam over-sedation and neurological damage in the intensive care unit by measuring 1-hydroxymidazolam glucuronide (1-OHMG), an active metabolite of midazolam in serum. 1-OHMG is known to have sedative properties [2,3]. Although the method described (i.e. high-performance liquid chromatography coupled to mass spectrometric detection) might be highly specific and sensitive to detect and quantify the presence of the 1-OHMG, the presence of an active substance irrespective of its quantity can never be solely indicative of a given (patho-)physiological response. Development of drug tolerance is a basic principle in clinical pharmacology, the benzodiazepines being a paradigmatic example. Making dose–response relationship assessment in vivo is thus very problematic. It is dangerous to rely on parameters (1-OHMG), even if very carefully measured, without considering the physiological component/response, as potentially important decisions could result from a diagnosis such as neurological damage in the setting of critical illness. In this regard, flumazenil, a selective benzodiazepine receptor antagonist, will remain indispensable.

Sufentanil in the ICU setting

3A4 enzyme system and is even a validated selective probe for P450 3A4 activity [4]; thus, the concomitant infusion of midazolam might have influenced the study results. Furthermore, Ethuin and colleagues [1] included “by chance” only male patitents in the study. Although controversial, gender may also affect pharmacokinetics of drugs metabolized by the CYP 450 3A4 system [3, 5]. In addition, the clearance of sufentanil is substantially dependent on changes in hepatic drug flow [7]. Factors determining hepatosplanchnic blood flow include the type and quantity of catecholamines applied [8], right cardiac hemodynamic situation as well as transpulmonary pressure. These data are not provided by the authors. All these factors might have contributed to the considerable inter-patient variation of the examined pharmacokinetic parameters. Nevertheless, the study by Ethuin and colleagues [1] shows a rapid decrease of plasma sufentanil concentration after prolonged infusion in the ICU setting. Further studies with a greater number of patients are needed to extrapolate these findings into clinical practice.