Escherichia coli ST117: exploring the zoonotic hypothesis.

Extraintestinal pathogenic Escherichia coli (ExPEC) can lead to severe infections, with additional risks of increasing antimicrobial resistance rates. Genotypic similarities between ExPEC and avian pathogenic E. coli (APEC) support a possible role for a poultry meat reservoir in human disease. Some genomic studies have been done on the ST117 lineage which contaminates poultry meat, carries multidrug resistance, can be found in the human intestinal microbiota, and causes human extraintestinal disease. This study analyzed the genomes of 61 E. coli from Brazilian poultry outbreaks focusing on ST117, to further define its possible zoonotic characteristics by genotypic and phylogenomic analyses, along with 1,699 worldwide ST117 isolates originating from human, animal, and environment sources. A predominance of ST117 was detected in the Brazilian isolates (n = 20/61) frequently carrying resistance to critical antibiotics (>86%) linked to IncFII, IncI1, or IncX4 replicons. High similarities were found between IncX4 from Brazilian outbreaks and those from E. coli recovered from imported Brazilian poultry meat and human clinical cases. The ST117 phylogeny showed non-specificity according to host and continent and an AMR index score indicated the highest resistance in Asia and South America, with the latter statistically more resistant and overrepresented with resistance to extended-spectrum beta-lactamases (ESBL). Most ST117 human isolates were predicted to have a poultry origin (93%, 138/148). In conclusion, poultry is a likely source for zoonotic ExPEC strains, particularly the ST117 lineage which can also serve as a reservoir for resistance determinants against critical antibiotics encoded on highly transmissible plasmids. IMPORTANCE: Certain extraintestinal pathogenic Escherichia coli (ExPEC) are particularly important as they affect humans and animals. Lineages, such as ST117, are predominant in poultry and frequent carriers of antibiotic resistance, presenting a risk to humans handling or ingesting poultry products. We analyzed ExPEC isolates causing outbreaks in Brazilian poultry, focusing on the ST117 as the most detected lineage. Genomic comparisons with international isolates from humans and animals were performed describing the potential zoonotic profile. The Brazilian ST117 isolates carried resistance determinants against critical antibiotics, mainly on plasmids, in some cases identical to those carried by international isolates. South American ST117 isolates from all sources generally exhibit more resistance, including to critical antibiotics, and worldwide, the vast majority of human isolates belonging to this lineage have a predicted poultry origin. As the world's largest poultry exporter, Brazil has an important role in developing strategies to prevent the dissemination of multidrug-resistant zoonotic ExPEC strains.

Nitric oxide homeostasis as a target for drug additives to cardioplegia.

The vascular endothelium of the coronary arteries has been identified as the important organ that locally regulates coronary perfusion and cardiac function by paracrine secretion of nitric oxide (NO) and vasoactive peptides. NO is constitutively produced in endothelial cells by endothelial nitric oxide synthase (eNOS). NO derived from this enzyme exerts important biological functions including vasodilatation, scavenging of superoxide and inhibition of platelet aggregation. Routine cardiac surgery or cardiologic interventions lead to a serious temporary or persistent disturbance in NO homeostasis. The clinical consequences are "endothelial dysfunction", leading to "myocardial dysfunction": no- or low-reflow phenomenon and temporary reduction of myocardial pump function. Uncoupling of eNOS (one electron transfer to molecular oxygen, the second substrate of eNOS) during ischemia-reperfusion due to diminished availability of L-arginine and/or tetrahydrobiopterin is even discussed as one major source of superoxide formation. Therefore maintenance of normal NO homeostasis seems to be an important factor protecting from ischemia/reperfusion (I/R) injury. Both, the clinical situations of cardioplegic arrest as well as hypothermic cardioplegic storage are followed by reperfusion. However, the presently used cardioplegic solutions to arrest and/or store the heart, thereby reducing myocardial oxygen consumption and metabolism, are designed to preserve myocytes mainly and not endothelial cells. This review will focus on possible drug additives to cardioplegia, which may help to maintain normal NO homeostasis after I/R.

Oxidatively modified plasma phospholipids containing reactive carbonyl functions measured by HPLC: evidence for phosphatidylcholine-bound aldehydes in plasma of burn patients.

A HPLC method has been developed to measure phosphatidylcholine (PC) containing reactive carbonyl functions in the sn-acyl residue in order to study processes in which such reactive carbonyls can be formed due to e.g. oxidative fragmentation. The method has been applied to determine PC-bound carbonyls as 2,4-dinitrophenylhydrazones (DNPH) in plasma of burn patients. Plasma from healthy volunteers served as controls. Additionally, in vitro oxidation experiments (A: plasma, buffer diluted; B: plasma + iron-EDTA complex and C: plasma + iron-EDTA complex + H2O2) have been performed to obtain and to identify 2,4-dinitrophenylhydrazine derivatizable carbonyl functions in plasma PC. Both, the PC-aldehydes and PC-aldehyde DNPH derivatives were cleavable with phospholipase C. Quantification was based on thin-layer chromatography purified soybean phosphatidylcholine, which was identically oxidized and derivatized as the plasma lipids in vitro.

The erythrocyte-perfused "working heart" model: hemodynamic and metabolic performance in comparison to crystalloid perfused hearts.

A brief period of ischemia was used to evaluate an erythrocyte-enriched Krebs-Henseleit (KH) buffer (n=8) compared to KH only (n=8) in an isolated working rabbit heart. Experimental protocol was as follows: preischemic baseline, 5 min of global ischemia followed by 45 min of reperfusion. Preischemic heart rate was identical, coronary flow was significantly lower (2.7 versus 5.6 mL/min/g wet wt, p<0.01), the other hemodynamic and biochemical values were significantly higher in erythrocyte-perfused hearts: aortic flow 23.5 versus 12.0, p<0.01; cardiac output 26.2 versus 17.6, p<0.01; all in mL/min/g wet wt; dp/dt max 1286 versus 997 mmHg/s, p<0.01; myocardial oxygen consumption 3.5 versus 2.3 micromol/min/g wet wt, p<0.05. During early reperfusion, in the erythrocyte-perfused hearts, coronary flow further increased (p<0.003), the other hemodynamic parameters returned to baseline values in both groups. High-energy phosphates showed significantly higher values (ATP 2.0+/-0.1 versus 1.3+/-0.1, p<0.05; CrP 2.0+/-0.2 versus 1.6+/-0.1, p<0.05 all in micromol/g wct wt), water content was significantly lower (81% versus 74%, p<0.05) in erythrocyte-perfused hearts. It can be concluded that the erythrocyte-perfused working heart model provides excellent oxygenation, leading to superior hemodynamic and metabolic performance. Additionally, in the erythrocyte-perfused hearts preservation of coronary flow reserve underlines the physiological competency of this preparation.

The cell in shock: the origin of multiple organ failure.

The immediate organ damage seen after multiple trauma and in shock is a typical example of non-bacterial inflammation triggered by activation of various mediators of both the humoral and cellular systems. Anaphylatoxins and the low-flow syndrome during the shock phase account for increased PMN* margination, which in turn causes pulmonary leukostasis and may provoke massive mediator release by PMN (oxygen radicals, proteinases, eicosanoids, PAF etc). This probably leads to severe endothelial cell damage, especially in the lung. Adherence of PMN to the endothelium appears to create the micro-environment where high concentrations of proteolytic enzymes and reactive oxygen radicals exert a deleterious effect on the cell membrane. Endothelial cell membrane injury leads to increased vascular permeability and cell edema. The development of the 'organ in shock' may require a few hours and initially cause minor or no functional impairment at all. Only when shock is severe is there early organ failure, which in this stage may still be an expression of non-bacterial inflammation. Numerous studies have reported the existence of shock-induced cardiodepressant substances in association with various forms of circulatory shock. We have determined a net negative inotropic effect of the low-molecular-weight plasma fraction in severe hypovolemic-traumatic shock and have isolated a cardiodepressant factor (CDF), which by blockade of the calcium inward current has a negative inotropic a chronotropic effect. The intestine as a shock organ appears to range first among the organs involved. The translocation of bacteria from the intestinal tract, the 'intestine in shock' represents the trigger reaction that eventually leads from the 'organ in shock', early organ failure to late (septic) organ failure. Here the most prominent factor is endotoxin (LPS) as a basic mediator of gram-negative bacteria, which also triggers the activation of humoral and cellular systems. The posttraumatic hyperdynamic phase commonly starts on days 3-5 and is mainly caused by bacteremia and/or endotoxemia. Macrophages have a major impact on the late phase of organ failure. At present, the most prominent cellular mediator of the lethal effect of endotoxin is thought to be cachectin, which is identical with the tumor necrotising factor (TNF). TNF is secreted by monocytes/macrophages (MO/MA) in response to LPS. Via macrophage derived cytokines and by LPS there is activation of endothelial cells, with increased adhesiveness for PMN. Both due to this increased adhesiveness and the presence of LPS and cytokines, PMN undergo massive activation, which causes mediator release and tissue damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Diltiazem during reperfusion preserves high energy phosphates by protection of mitochondrial integrity.

OBJECTIVE: This study evaluates the effects of diltiazem administered during reperfusion on hemodynamic, metabolic, and ultrastructural postischemic outcome. METHODS: Hearts of 38 adult White New Zealand rabbits underwent 60 min of global cold ischemia followed by 40 min of reperfusion in an erythrocyte perfused isolated working heart model. Hearts were randomly assigned to four groups and received diltiazem (0.1, 0.25, and 0.5 micromol/l) during reperfusion only, or served as control. RESULTS: The postischemic time courses of heart rate, aortic flow, and external stroke work clearly reflected the dose-dependent negative chronotropic and inotropic efficacy of diltiazem in the two higher concentrations. High energy phosphates (HEP) determined from myocardial biopsies taken after 40 min of reperfusion were significantly better preserved in all treatment groups compared to control hearts. Similarly ultrastructural grading of mitochondria and myofilaments revealed a significant reduction of reperfusion injury in hearts that received diltiazem compared to control. CONCLUSIONS: Diltiazem protects mitochondrial integrity and function, thereby preserving myocardial HEP levels. Only low dose diltiazem (0.1 micromol/l) during reperfusion combines both, optimal mitochondrial preservation with minimal changes in hemodynamics.

S-nitroso albumin enhances bone formation in a rabbit calvaria model.

Nitric oxide (NO) is a mediator involved in bone regeneration. We therefore examined the effect of the novel NO donor, S-nitroso human serum albumin (S-NO-HSA) on bone formation in a rabbit calvaria augmentation model. Circular grooves (8 mm diameter, two per animal) were created by a trephine drill in the cortical bone of 40 rabbits and titanium caps were placed on the rabbit calvaria bone filled with a collagen sponge soaked with either 100 µL S-NO-HSA (5%, 20%) or human albumin (5%, 20%). After 4 weeks the titanium hemispheres were subjected to histological and histomorphometric analysis. Bone formation and the volume of the residual collagen sponge were evaluated. S-NO-HSA treatment groups had a significantly higher volume of newly formed bone underneath the titanium hemispheres compared to the albumin control groups (5%: 15.5 ± 4.0% versus 10.6 ± 2.9%; P < 0.05; 20%: 14.0 ± 4.6% versus 6.0 ± 3.8%; P < 0.01). The volume of residual collagen sponge was also significantly lower in the S-NO-HSA groups compared to the control groups (5%: 0.4 ± 0.5% versus 2.6 ± 2.4%; P < 0.05 and 20%: 1.5 ± 2.7% versus 13.0 ± 18.7%; P < 0.01). This study demonstrates for the first time that S-NO-HSA promotes bone formation by slow NO release. Additionally, S-NO-HSA increases collagen sponge degradation.

New HTK-N46B cardioplegia provides superior protection during ischemia/reperfusion in failing hearts.

AIM: The aim of this paper was to improve operative outcome during open-heart surgery in patients with failing hearts, the composition of cardioplegic solutions has to be further optimized. HTK-N46b, a novel cardioplegic solution, has been developed for efficient protection of the energy state of myocytes as well as endothelial cells. Aim of this study is the evaluation of HTK-N46b in comparison to its precursor Custodiol® (HTK) in failing rat hearts undergoing ischemia/reperfusion. METHODS: In male Sprague Dawley rats myocardial infarction (MI) was induced by LAD ligation. Six weeks after MI cardiac function was determined by transthoracic echocardiography. Sixteen animals with hearts showing a fractional shortening <25% were randomly assigned to two groups, HTK (N.=8) and HTK-N46b (N.=8). After excision hearts were evaluated in an erythrocyte-perfused isolated working heart model. Cold ischemia (4°C) for 60 minutes was followed by 45 minutes of reperfusion. Cardiac arrest was induced either with HTK or HTK-N46b at the beginning of ischemia. RESULTS: At similar preischemic fractional shortening (HTK-N46b: 14.41±1.83% vs. HTK: 14.91±1.92%; NS) postischemic recovery of stroke volume and stroke work were significantly improved in the HTK-N46b rat hearts compared to HTK. Concerning recovery of coronary flow there was no difference between groups. At the end of reperfusion the HTK-N46b protected group revealed higher levels of ATP (HTK-N46b: 22.01±0.89 nmol/mg protein vs. HTK: 16.83±1.72 nmol/mg protein; P<0.05) and energy charge (HTK-N46b: 0.82±0.02 vs. HTK: 0.74±0.02; P<0.05). CONCLUSION: HTK-N46b showed superior cardioprotective properties according to postischemic hemodynamic recovery and biochemical markers compared to HTK in failing rat hearts.

S-nitroso human serum albumin given after LPS challenge reduces acute lung injury and prolongs survival in a rat model of endotoxemia.

Endotoxemia leads to the induction of inducible nitric oxide synthase (NOS-2) and increased expression of numerous inflammatory mediators contributing to endotoxin-induced acute lung injury. We tested the hypothesis that supplementation of nitric oxide (NO) by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) given after lipopolysaccharide (LPS) challenge may reduce NOS-2 expression, lung inflammation and acute lung injury. Rats were divided into four groups: sham-operated (no treatment), LPS, LPS+HSA (human serum albumin), and LPS+S-NO-HSA. LPS was administered intravenously (20 mg kg(-1)) resulting in acute lung injury and a high mortality rate within 6 h (>90%). LPS-induced lung injury was characterized by an increased lung edema (lung wet/dry weight ratio), pulmonary neutrophil infiltration (myeloperoxidase activity, MPO) as well as a robust inflammatory response [increased expression of intercellular adhesion molecule-1 (ICAM-1), NOS-2, and cyclooxygenase-2 (COX-2)]. Infusion of S-NO-HSA or HSA was started 2 h after LPS and continued for 4 h (total dose of 72 mg kg(-1)) at a rate of 300 microg kg(-1) min(-1). S-NO-HSA but not HSA prolonged survival of endotoxemic rats, reduced the hypotensive response to LPS, minimized LPS-induced lung edema and injury, normalized MPO activity as well as diminished lung expression of pro-inflammatory molecules such as ICAM-1, NOS-2, and COX-2. Continuous supplementation of NO by S-NO-HSA after LPS challenge prevents induction of NOS-2, provides significant protection of endotoxin-induced acute lung injury, and prevents early mortality in endotoxic shock in rats. Our results suggest a potential therapeutic role for S-NO-HSA in endotoxemia.

Role of Toll-like receptors 2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis.

Neutrophil dysfunction in alcoholic hepatitis is associated with endotoxemia and an increased incidence of infection, but the mechanism is unclear. We aimed to investigate the role of Toll-like-receptors (TLR)2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis. Neutrophils from healthy volunteers were incubated with alcoholic hepatitis patients' plasma (n = 12) with and without TLR2, 4, or 9 antagonists and with and without human albumin. TLR2, 4, and 9 expression, neutrophil oxidative burst, phagocytosis, and CXCR1+2 expression were measured by FACS analysis. Patients' plasma increased oxidative burst, decreased CXCR1+2 expression, and decreased phagocytosis of normal neutrophils in association with increased expression of TLR2, 4, and 9 and depletion of ATP. Inhibition of TLR2, 4, and 9 prevented the increase in oxidative burst and the decrease in CXCR1 and CXCR2 expression but did not prevent phagocytic dysfunction. Incubation with albumin completely prevented the patient plasma induced neutrophil dysfunction. Increased expression of TLR2, 4, and 9 is associated with neutrophil dysfunction, endotoxemia, and energy depletion. TLR2, 4, and 9 inhibition does not improve phagocytosis, indicating that TLR overexpression may be the result and not the cause of neutrophil activation. Albumin, an endotoxin scavenger, prevents the deleterious effect of patients' plasma on neutrophil phagocytosis, resting burst, and TLR expression.

Simultaneous determination of myocardial nucleotides, nucleosides, purine bases and creatine phosphate by ion-pair high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromatographic method is described for the separation and quantification of myocardial nucleotides, nucleosides, their metabolites and creatine phosphate-related compounds in a single run. Separation of a standard mixture containing 21 compounds was achieved on a 5-microns Hypersil ODS column with a 5-min isocratic elution (buffer: 0.1 M NaH2PO4, pH 5.5, containing 5.9 mM tetrabutylammonium hydrogen-sulphate) followed by a slow linear gradient to 17% acetonitrile. The method was applied to extracts of freeze-clamped rat heart tissue samples as well as to extracts of neonatal rat heart cardiomyocytes, and it provided good resolution of high-energy phosphates, including creatine phosphate, as well as of their degradation products.

Inhibition of human, ovine, and baboon neutrophil elastase with Eglin c and secretory leukocyte proteinase inhibitor.

The association rate constants (kon) of human, ovine, and baboon neutrophil elastase with two recombinant serine proteinase inhibitors (Eglin c, secretory leukocyte proteinase inhibitor) were compared. The association rate constant of sheep leukocyte elastase (SLE) with Eglin c is about 100 times lower (kon = 2.2 x 10(5) M-1s-1) than that of human elastase (kon = 2.4 x 10(7) M-1s-1). Baboon elastase, however, is as effectively blocked with Eglin c (kon = 2.5 x 10(7) M-1s-1) as human elastase. Secretory leukocyte proteinase inhibitor (SLPI) blocks the elastase of all three species with high efficiency; baboon elastase shows the highest association rate constant (kon = 5.6 x 10(7) M-1s-1) followed by human elastase (kon = 4.1 x 10(7) M-1s-1) and finally sheep elastase (kon = 1.2 x 10(7) M-1s-1). These findings demonstrate marked differences in the inhibition kinetic properties of ovine and human elastase. Concerning a future clinical application of proteinase inhibitors, the baboon seems a more suitable model than sheep to evaluate the effects of Eglin c and SLPI, since both inhibitors block baboon and human elastase with comparable efficiency.

Net inotropic plasma activity in canine hypovolemic traumatic shock: low molecular weight plasma fraction after prolonged hypotension depresses cardiac muscle performance in vitro.

Numerous studies report controversial results about the occurrence and role of cardiodepressant substances in various forms of circulatory shock. We investigated the net inotropic effect of the low molecular weight fraction (mol wt less than or equal to 1,000) of plasma in prolonged canine hypovolemic traumatic shock using an in vitro guinea pig papillary muscle assay (isotonic mode). The shock plasma fractions (ultrafiltrates) after 4 hr of hypotension (mean arterial blood pressure 40-50 mm Hg) and immediately post-reinfusion significantly depressed papillary muscle function (P less than .02). The extent of papillary muscle shortening was decreased by 49.5 +/- 9.9% in pre- and 50.6 +/- 10.0% in post-reinfusion plasma ultrafiltrates (mean values +/- standard error of the mean; n = 6 shock experiments). In contrast, both the plasma ultrafiltrates from ten non-anesthetized healthy dogs and the control ultrafiltrates obtained prior to onset of shock in the experiments (-6.4 +/- 2.6; n = 6) induced no significant change of the in vitro performance of papillary muscle contraction. These results were achieved with plasma fractions in which ionized calcium and pH were adjusted to concentrations equivalent to the bioassay solution. Lactate acidosis and severe hypoglycemia (1.97 +/- 0.43 mM post-reinfusion) occurred in the shock experiments. Lack of energy substrate (glucose) was not responsible for the in vitro depression. Four depressive shock ultrafiltrates with glucose concentrations adjusted to control ultrafiltrate levels induced a 66.6 +/- 8.8% decrease in the extent of papillary muscle shortening. These results suggest that the possible occurrence of high net negative inotropic activity in plasma, especially just post-reinfusion, may play a role in the pathogenesis of irreversible circulatory shock.

Preliminary data on isolation of an elastase-like proteinase and its inhibitor from ovine neutrophil granulocytes.

Ovine polymorphonuclear leukocytes (PMNs) were purified by counterflow centrifugation of the buffy coat. The resulting cell preparation was gently sonicated and ultracentrifuged for separation of cytosol and cell organelles. The proteolytic activity of the isolated enzyme was determined with the specific substrate MeO-Suc-Ala-Ala-Pro-Val-Nan (methoxysuccinyl-[L-alanyl]2-L-prolyl-L-valine p-nitroanilide). Elastase-like activity was identified in the insoluble (particular) fraction, while an inhibitor of elastase activity was found in the supernatant (cytosolic fraction). Further purification of the elastase-like enzyme was achieved by cation exchange chromatography (SP-Sephadex C-25 and CM-Sephadex C-25) and gel permeation chromatography (Bio-Gel P-30). Purification of the inhibitor from cytosol was accomplished using anion exchange chromatography (DE32 diethylaminoethyl cellulose) and two gel permeation steps (Sephadex G-75 and TSK-G 2000 SW - HPLC column). Degrees of purity in all separation steps were controlled with SDS-polyacrylamide gel electrophoresis. The molecular mass of the elastase-like enzyme is in a range of 25 to 27 kDa. The isoelectric point is between 8.0 and 9.0. Three isoenzymes were found. The optimal activity lies at pH 8.0. The molecular mass of the inhibitor is about 40 kDa.

The enzymatic and release characteristics of sheep neutrophil elastase: a comparison with human neutrophil elastase.

Sheep are often used to study tissue damage following shock after traumatic injury and in the course of other diseases. The processes involved are thought to be caused at least in part by elastase released from polymorphonuclear leukocytes (PMNs). Since little is known about elastase and its role as a mediator of tissue damage in sheep, we studied the biochemical properties and release characteristics to sheep leukocyte elastase (SLE) in comparison of those of human leukocyte elastase (HLE). Both enzymes showed similar molecular masses, amino-acid compositions, N-terminal amino-acid sequences, and abilities to digest elastin substrates. Differences, however, were found in kinetic parameters measured with the elastase-specific substrate N-methoxysuccinyl-(L-alanyl)2-L-prolyl-L- valine-4-nitroanilide (MeoSuc-AAPV-pNa). The Michaelis constant (Km) of ovine elastase was nearly 10 times higher (1.82 mM) than the Km of HLE (0.21 mM). Values of SLE calculated for kcat were 70% and for kcat/Km 8% of corresponding values determined for HLE. In addition, significant differences between sheep and human PMNs were found in in vitro stimulation experiments. In contrast to human PMNs, sheep neutrophils released no active elastase, and only 50 to 70% of the H2O2 produced by human PMNs. This failure to release active elastase could not be explained by a lower elastase content of sheep PMNs, as there were no significant differences found between the elastase contents of sheep and human PMNs. We conclude that elastase liberated by stimulated sheep PMNs is inactivated by a concomitantly released proteinase inhibitor also located within the sheep PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperdynamic sepsis in baboons: I. Aspects of hemodynamics.

The baboon has a number of advantages as a shock model, as its physiological as well as its biochemical behaviour is similar to man. Therefore we have tried to set up a model to mimic the early hyperdynamic phase of clinical sepsis. Seven baboons, 21-25 kg body weight were kept under EEG servocontrolled anesthesia for 8 hr. During this time live E. coli (ATC #33985) 2 x 10(10) BW/8 hr were continuously infused intravenously. Adequate fluid supply with Ringer's solution (up to 40 ml/kg/hr) was given to keep the pulmonary artery wedge pressure at baseline levels; this procedure resulted in a hyperdynamic response with a cardiac output (CO) 20-35% above baseline and a decrease (20-39%) in mean arterial pressure (MAP), leading to a 50% decrease in peripheral resistance. The pulmonary vascular changes were reflected in an increase of the mean pulmonary pressure (PA) to 42% above baseline and a marked rise in pulmonary vascular resistance (PVR) to 50% above baseline with no additional changes in pulmonary gas exchange. After 8 hr both CO and MAP were inversely correlated (r = 0.9-1) with dramatically increased catecholamine plasma levels (15 times above baseline). With continuous infusion of live E. coli (blood levels 10(5)-10(6) CFU/ml) and massive fluid supply we have successfully mimicked hyperdynamic sepsis with severe organ failure after an 8-hr observation period.

Mechanisms in acute septic cardiomyopathy: evidence from isolated myocytes.

Although often not considered, the heart is one of the targets of multiple organ failure in sepsis and septic shock, with myocardial depression being a prominent component of this "acute septic cardiomyopathy". Hypotheses concerning the etiology of this depression are increasingly elucidated on a cellular level, including dysfunction of the beta-adrenoceptor/G protein/adenylate cyclase system, calcium channel blockade by cardiodepressant factor, contractile impairment by activated leucocytes, as well as inhibition of protein synthesis by Pseudomonas exotoxin A. In the search for "mechanisms of myocardial depression in sepsis", isolated cardiomyocytes may play a role as research tools with respect to: a) discrimination between direct and indirect cardiodepressant effects; b) identifying not only the acute, but also chronic toxin- and mediator-induced cardiodepression; c) clarification of the mechanism of action of cardiodepressant bacterial toxins and sepsis mediators; d) establishment of in vitro models of leucocyte-mediated cardiodepression in sepsis.

A cardiodepressant factor isolated from blood blocks Ca2+ current in cardiomyocytes.

A cardiodepressant factor (CDF) was isolated (salt free) from the plasma of dogs after hypovolemic-traumatic shock by column chromatography. CDF was found to exert a concentration-dependent negative inotropic effect in guinea pig papillary muscle; it reduced the amplitude of cell wall motion, the contraction and relaxation velocity, and the frequency of spontaneously beating rat cardiomyocytes in culture, effects that were rapidly reversible upon washout. By means of electrophysiological methods (whole cell recording with patch electrodes and voltage clamp) we tested the effect of CDF on adult guinea pig cardiomyocytes: application of CDF initially decreased the plateau by 7 mV (150 ms after peak of action potential) and reduced the action potential duration by 93 ms (76% of control action potential duration) at 50% and 88 ms (79%) at 90% of repolarization. The plateau was further reduced by 13 mV and the action potential duration was prolonged by 25 ms (106%) at 50% and was prolonged drastically by 156 ms (137%) at 90% of repolarization compared with control. Voltage-clamp experiments have shown that the most prominent effect of CDF is a strong reduction of ICa accompanied by inhibition of IK and subsequent repolarization. Similar results have been obtained with neonatal rat cardiomyocytes. Blockage of the calcium inward current can explain the negative inotropic and chronotropic effect of CDF in cardiomyocytes.