Klaus Tschaikowsky

Coincidence of pro- and anti-inflammatory responses in the early phase of severe sepsis: Longitudinal study of mononuclear histocompatibility leukocyte antigen-DR expression, procalcitonin, C-reactive protein, and changes in T-cell subsets in septic and postoperative patients.

ObjectiveTo determine the time course of histocompatibility leukocyte antigen (HLA)-DR expression in peripheral blood mononuclear cells and their relationship to markers of inflammation, organ function, and outcome during severe sepsis. DesignProspective, longitudinal study. SettingUniversity hospital intensive care unit. PatientsTwenty-three postoperative patients with severe sepsis and 26 patients with uneventful postoperative course as well as 24 healthy, age-matched subjects. InterventionsSerum procalcitonin was determined by using an immunochemiluminescence assay, and C-reactive protein and leukocyte antigens were determined by using flow cytometry over 14 days in parallel with clinical data collection. Measurements and Main ResultsDespite a relative lymphopenia, absolute lymphocyte counts and CD4+/CD8+ T-cell ratio in septic patients were significantly elevated above normal. Particularly, CD4+ and CD8+ T-cell counts in nonsurvivors of sepsis were approximately twice as high as those of survivors. Significantly decreased monocytic HLA-DR expression was observed in both survivors and nonsurvivors at the onset of severe sepsis. Percentages of HLA-DR+ lymphocytes, however, were significantly increased during sepsis, especially in nonsurvivors. Whereas survivors of sepsis showed a continuous recovery of monocytic HLA-DR expression to ≥70% within 10 days, nonsurvivors were characterized by a second decrease in monocytic HLA-DR expression after day 7 or a permanent suppression (<40%). Peak of systemic inflammatory reaction, documented by maximum serum concentrations of procalcitonin and C-reactive protein, coincided with the nadir of monocytic HLA-DR expression. Moreover, procalcitonin and C-reactive protein as well as scores on the Acute Physiology and Chronic Health Evaluation II and Sepsis Organ Failure Assessment were inversely correlated with the monocytic HLA-DR expression. ConclusionsDecreases in monocytic HLA-DR expression occurred simultaneously with signs of hyperinflammation as early as the onset of severe sepsis and usually developed in opposite directions than inflammatory markers and sepsis severity scores.

Fish oil-supplemented parenteral diets normalize splanchnic blood flow and improve killing of translocated bacteria in a low-dose endotoxin rat model.

Objective: Intestinal ischemia decreases barrier function of the gut and enhances translocation of bacteria and toxins. Several studies indicate that fish oil can modulate prostaglandin formation and thus, regional blood flow and immune function. This study was performed to determine the effects of parenteral diets with omega‐3 fatty acids on microcirculation and barrier function of the gut. Design: Prospective, randomized, controlled animal study. Setting: University laboratory. Subjects: A total of 64 male Sprague‐Dawley CD rats. Interventions and Measurements: For 48 hrs, eight groups of eight rats each received total parenteral nutrition with four different types of lipids. The source of fat in group L was soybean oil only and in group L‐M a mixture of soybean oil and medium‐chain triglycerides. In groups FO‐20 and FO‐40, 20% or 40%, respectively, of the soybean oil in group L‐M was replaced by fish oil. The other four groups received an additional continuous infusion of endotoxin (0.1 mg/100 g body weight per day) for the last 24 hrs. Blood flow was measured with microspheres, and translocation was determined by microbiological methods and instillation of radioactive‐marked bacteria into the gut. Main Results: In the animals without fish oil, the endotoxin application reduced the blood flow to the intestine ∼25%. Animals with fish oil in their diets showed normal values. Translocation of gut bacteria was increased significantly in all endotoxin groups. However, less‐viable bacteria could be detected in the animals with fish oil diets in their mesenteric lymph nodes and livers. Conclusions: In this model, diets enriched with fish oil abolish the endotoxin‐induced decrease of nutritive blood flow to the gut and ameliorate the bactericidal defense of the splanchnic region. The lower count of viable bacteria in the fish oil groups is more related to an improved killing of translocated bacteria than a reduction of the translocation rate.

Procalcitonin - Influence of temperature, storage, anticoagulation and arterial or venous asservation of blood samples on procalcitonin concentrations

In this study we have analysed the influence of temperature and time of storage and of repeated freezing on procalcitonin plasma concentrations ex vivo. We have also analysed the difference of procalcitonin concentrations in arterial or venous blood samples and the influence of different anticoagulation techniques on procalcitonin concentrations (serum, EDTA-, lithium-heparin- or citrate plasma). At room temperature (25 degrees C) a loss of procalcitonin plasma concentrations of 6.4% +/- 2.6% (mean, 2 standard error of the mean) after 3 hours (4.6% +/- 5.2% at 4 degrees C) and 12.3% +/- 3.1% after 24 hours occurred (6.3% +/- 5.0% at 4 degrees C, n = 17 each). Comparing the procalcitonin concentrations of blood samples with different anticoagulants (n = 24 each), there was only a significant difference between procalcitonin concentrations in heparinized plasma and serum (+ 7.6%, difference of the mean). There was no significant influence of the blood sampling technique (arterial or venous line) and of repeated freezing/thawing cycles (up to 3 times) on the procalcitonin concentrations measured. Although the difference of sampling and storage of the blood on procalcitonin concentrations is not significant, multiple factors may act synergistically on the result of procalcitonin measurement. To keep variations of ex vivo conditions as minimal as possible, a standardized technique of anticoagulation, time and temperature of storage is recommended, e.g. the use of EDTA-plasma and storage at room temperature, when samples are measured within 4 hours after blood drawing.

Endothelin in septic patients: effects on cardiovascular and renal function and its relationship to proinflammatory cytokines.

Objective To determine the time course of big-endothelin (big-ET) and its relationship to proinflammatory cytokines and organ function in sepsis. Design Prospective analysis in patients meeting criteria of severe sepsis as part of a multicenter study (RAMSES) with an anti-tumor necrosis factor monoclonal antibody F(ab′)2 fragment (afelimomab). Setting University hospital intensive care unit. Patients A total of 23 nontrauma patients with severe sepsis or septic shock and ten multiple trauma patients. Septic patients were randomized for additional experimental treatment when initial interleukin (IL)-6 serum level was above 1000 pg/mL. Interventions Randomized patients received 1.0 mg/kg afelimomab or placebo three times daily over 3 days in addition to standard treatment. In each patient, serial blood samples for plasma big-ET and cytokine determination as well as clinical data were collected over 28 days. Measurements and Main Results Significantly increased concentrations of circulating big-ET were found in patients with severe sepsis as compared with healthy subjects. In septic patients, big-ET plasma levels were higher than in multiple trauma patients, and were more elevated in randomized than in nonrandomized patients. At study entry (day 0), big-ET reached a peak concentration and significantly correlated with IL-6 (r2 = .43) and IL-8 (r2 = .44) in patients with severe sepsis. Moreover, big-ET levels in septic patients, pooled over all observation days, correlated positively with pressure-adjusted heart rate, central venous pressure, pulmonary artery pressure, and pulmonary vascular resistance and correlated inversely with creatinine clearance (r2 = .54, .54, .59, .40, and .51, respectively, p = .0001). In all randomized septic patients, pressure-adjusted heart rate decreased from day 0 to day 2 in parallel with big-ET; however, a significant decrease in big-ET (day 0 to day 2) was only found in patients additionally treated with afelimomab. Conclusions In patients with severe sepsis, big-ET plasma levels are markedly increased, even above those of multiple trauma patients, in close relationship to IL-6 and IL-8, and with significant correlation to renal function and pulmonary vascular tone.

Blood volume determination using hydroxyethyl starch: a rapid and simple intravenous injection method.

OBJECTIVE To develop and evaluate a new method for blood volume measurements using hydroxyethyl starch as a dilution marker. DESIGN Laboratory and clinical investigation. SETTING Neurosurgical operating rooms and anesthesiological laboratories of a university hospital. PATIENTS Twelve patients who underwent a neurosurgical operation. INTERVENTIONS Anesthesia and operations were carried out by physicians who were not involved in the study. In addition, blood samples were drawn from 50 volunteers. MEASUREMENTS AND MAIN RESULTS Blood volume measurements by the hydroxyethyl starch method were validated in vivo by comparison with a conventional carbon monoxide technique. Patients were intravenously injected with hydroxyethyl starch (100 mL) and received simultaneously an injection of carbon monoxide (50 mL) into a closed-circuit ventilation system. Blood samples obtained before and 5 mins after injection were analyzed for carboxyhemoglobin and glucose plasma concentrations after acidic hydrolysis of hydroxyethyl starch. Blood volume was calculated from the difference between glucose concentrations measured after hydrolysis in the plasma, before and after the addition of hydroxyethyl starch. In vitro, the hydroxyethyl starch method had an error and a precision of approximately 2%. In vivo, simultaneous measurements of blood volume using hydroxyethyl starch and carbon monoxide demonstrated a high correlation (r2 = .96, p < .001) between these methods. The mean difference between the two methods relative to their average value was 1.0 +/- 3.5%; the bias was 52.3 mL, and the 95% confidence interval was -64.0 to +168.7 mL. CONCLUSIONS Blood volume determination by the hydroxyethyl starch method is accurate and rapid and may enhance perioperative monitoring of fluid and blood therapy.

Effects of liposome-encapsulated dichloromethylene diphosphonate on macrophage function and endotoxin-induced mortality

Dichloromethylene diphosphonate (Cl2MDP), encapsulated into liposomes, is known to eliminate splenic and hepatic macrophages. In the present study we investigated the time course of the effects of i.v. injected Cl2MDP-liposomes on macrophage function in rats from 2 h to 14 days postinjection. We further assessed the impact of Cl2MDP-liposomes on endotoxin-induced lethality. Magnetometry was used to measure uptake and distribution of magnetic particles in different organs and to non-invasively monitor cell organelle motion and disappearance of particles in Kupffer cells. Cl2MDP-liposomes administered to rats pre-injected with magnetic particles, resulted in a biphasic impairment of the cell organelle motion after 1 h and 48 h. At 8 h, retained particles started to leave the liver, indicating the begin of an irreversible damage to hepatic macrophages. The released magnetic material was redistributed and taken up primarily by spleen and bone marrow. Pretreatment with Cl2MDP-liposomes for 24 h and 48 h significantly depressed phagocytic capacity for magnetic particles in Kupffer cells. Again, this was compensated by an increased uptake by spleen, lungs, and bone marrow. Interestingly, i.v. administration of endotoxin after pretreatment with Cl2MDP-liposomes resulted in a significant reduction in mortality from 55% to 14%.

Protein kinase C inhibitors suppress LPS-induced TNF production in alveolar macrophages and in whole blood: The role of encapsulation into liposomes

Tumor necrosis factor (TNF) is a pivotal mediator of endotoxin shock, but the regulation of lipopolysaccharide (LPS)-induced TNF production in different populations of mononuclear cells has not been fully clarified. Protein kinase C (PKC) is thought to play a central role in signal transduction in response to inflammatory stimuli. We studied the effect of two PKC inhibitors, staurosporine (STP) and sphingosine (SPG), on TNF production in rat alveolar macrophages (AM) and in whole blood (BM) incubated with 0.25-25,000 ng/ml of LPS. We also assessed the role of STP encapsulation into pH-sensitive and pH-insensitive liposomes composed of cholesterolhemisuccinat/dioleoylphosphatidyl ethanolamine and cholesterolhemisuccinat/distearylphosphatidyl choline, respectively. LPS induced a dose-dependent TNF response that was 2.5-4.5-times higher in AM than in BM with the same amount of monocytes. SPG and STP significantly reduced TNF in both cultures by 40-96%. Encapsulation of STP into pH-sensitive, but not pH-insensitive liposomes, significantly increased the effectiveness of TNF suppression. We conclude that the LPS-induced TNF production by AM and BM is strongly dependent on PKC activation. However, AM were less sensitive to PKC inhibition than BM.

Staurosporine encapsulated into pH-sensitive liposomes reduces tnf production and increases survival in rat endotoxin shock.

Bacterial lipopolysaccharide (LPS) can elicit septic shock; however, there is growing evidence that most of its pathophysiological effects are mediated by the release of tumor necrosis factor (TNF) and other cytokines. In turn, LPS-induced TNF production is thought to implicate the activation of intracellular protein kinase C (PKC). In this study, we examined whether pH-sensitive liposomes containing staurosporine (STP), a potent inhibitor of PKC, when injected intravenously would suppress TNF production and reduce mortality in an endotoxin rat model. We found that pretreatment of rats with pH-sensitive STP-liposomes by intravenous administration 1.5 h prior to LPS injection decreased lethality from 80% to approximately 30%. Importantly, this improvement in outcome was associated with significant reductions in TNF serum levels; 1 h after LPS injection serum TNF was 73% lower than in a saline control group, and at 2 h TNF levels were 84% lower. STP-liposome pretreatment also ameliorated the severe reduction in body temperature, characteristic for a hypodynamic shock, that was observed in LPS-challenged rats, but had relatively little effect on the transient leukopenia. We conclude that STP-liposomes can suppress LPS-induced TNF production by the mononuclear phagocytic system, can reduce the symptoms of septic shock, and can increase survival.