Dieter Fröhlich

Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specific genetic predisposition to sepsis.

ObjectivesTo determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 → Glu;Pst I polymorphism in intron 5; silent mutation G545 → C) and the lipopolysaccharide binding protein (LBP) (Cys98 → Gly; Pro436 → Leu) are associated with the incidence and lethality of sepsis. DesignCase control study of patients with sepsis. SettingIntensive care units within university hospitals. PatientsA total of 204 patients diagnosed with sepsis and 250 healthy blood donors. InterventionsNone. Measurements and Main Results Short DNA fragments containing the polymorphic sites of the LBP and BPI locus were amplified by the polymerase chain reaction or mismatched polymerase chain reaction. The individual polymorphisms were determined with the appropriate restriction enzyme digestions and subsequent agarose gel electrophoresis. The presence of LBP genotypes with the less frequent Gly98 allele was found to be associated with sepsis (p < .02) in male patients, but not in females. Patients which were homozygote for either of the rare Gly98 (n = 6) and/or Leu436 (n = 5) LBP alleles, furthermore, exclusively were nonsurvivors of sepsis. The genotype frequencies in the BPI gene did not differ between patients and control individuals. ConclusionsOur findings suggest that common polymorphisms in the gene for LBP in combination with male gender are associated with an increased risk for the development of sepsis and, furthermore, may be linked to an unfavorable outcome. These data support the important immunomodulatory role of LBP in Gram-negative sepsis and suggest that genetic testing may be helpful for the identification of patients with an unfavorable response to Gram-negative infection.

Cytokine upregulation of surface antigens correlates to the priming of the neutrophil oxidative burst response

Neutrophil activation is strongly related to organ dysfunction that occurs during systemic inflammatory responses. The aim of our study was to analyze the oxidative burst response in correlation to the up‐ and downregulation of N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine (fMLP) receptors and the surface antigens CD11b, CD62L, and CD66b as potential surrogate markers of the degree of neutrophil priming for an increased oxidative burst response induced by proinflammatory cytokines.

Changes in HDL-associated apolipoproteins relate to mortality in human sepsis and correlate to monocyte and platelet activation.

ObjectiveLipoproteins modulate vascular cell function in inflammation. In this study, we analyzed whether plasma concentrations of lipoproteins and apolipoproteins in human sepsis are related to patient survival and the activation of blood monocytes and platelets.DesignObservational study.SettingMedical and surgical intensive care units (ICU) of a university hospital.Patients151 consecutive patients after sepsis criteria had been met for the first time.InterventionsNone.MeasurementsPlasma lipoproteins, apolipoproteins, platelet CD62P-expression, monocyte HLA-DR-expression, SAPS II-scores (Simplified Acute Physiology Score) and 30-day-mortality were recorded.ResultsTotal cholesterol, high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) cholesterol, apolipoprotein (apo)-AI and apo-B were all found to be significantly lower in non-survivors than in survivors. In contrast to other (apo)lipoproteins, apo-AI and HDL cholesterol further decreased in non-survivors during the ICU stay. Logistic regression analysis revealed apo-AI to be an independent predictor of 30-day-mortality. A significant inverse correlation was found for apo-AI/HDL-cholesterol and platelet activation. Later in the course of the disease, HLA-DR expression on monocytes correlated positively to apo-AI and apo-CI concentrations and inversely to the apo-E concentration.ConclusionLow apo-AI is independently related to 30-day mortality in human sepsis and the decrease in apo-AI/HDL cholesterol correlates to increased platelet activation. Moreover, changes in apolipoproteins supposed to modulate lipopolysaccharide effects, such as apo-CI and apo-E, correlate to monocyte activation.

Mild hyperthermia down-regulates receptor-dependent neutrophil function

Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of unopsonized bacteria. We evaluated various functions at 33°–41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecule expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature-independent. However, up-regulation of CD11b with tumor necrosis factor (TNF)-&agr; was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature-dependent. Bacterial uptake was inversely related to temperature, more so with Escherichia coli than Staphylococcus aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-formyl-l-methionyl-l-leucyl-phenylalanine receptors on neutrophils: hyperthermia decreased expression, especially with TNF-&agr;. N-formyl-l-methionyl-l-leucyl-phenylalanine-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-&agr;. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to nonreceptor-dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated.

Nitrous oxide impairs the neutrophil oxidative response

Background Nitrous oxide has been shown inconsistently to impair the oxidative function of neutrophils. The choice of the stimulus, receptor agonists, or stimuli acting independent of receptors seems to determine whether nitrous oxide impairs the oxidative functions, suggesting an interference with the cytosolic signaling of neutrophils. Methods Production of hydrogen peroxide by neutrophils was assessed using flow cytometric analysis. N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), C5a, dioctanylglycerol, and phorbol‐12‐myristate‐13‐acetate were used as stimuli. In addition, the expression of receptors for FMLP and the cytosolic‐free calcium response of cells were measured. Results Nitrous oxide depresses C5a‐ or FMLP‐induced generation of reactive oxygen derivatives in a concentration‐dependent manner. However, the response with direct activation of protein kinase C was unaffected. Further, the number of FMLP receptors and the cytosolic calcium response were unaffected. Inhibition of the oxidative response was not reversible within the observation period of 4 h. Conclusions Nitrous oxide inhibited the intracellular signaling of the investigated G‐protein‐coupled receptors for chemotactic peptides. No interference of nitrous oxide with reduced nicotinamide adenine dinucleotide phosphate oxidase, the oxidative enzyme system of neutrophils, nor with its activation through protein kinase C was detected.

Volatile anaesthetics induce changes in the expression of P-selectin and glycoprotein Ib on the surface of platelets in vitro

Halothane has been shown to inhibit platelet aggregation and may, therefore, prevent intraoperative platelet activation. The aim of this study was to compare the effects of volatile anaesthetics on platelet activation, including the transformation into an adhesive platelet phenotype. After in vitro exposure to volatile anaesthetics, the expression of the adhesion molecule P-selectin and the internalization of the receptor for the von Willebrand factor (GPlb) were analysed by flow cytometry. In contrast to desflurane or N2O, sevoflurane (> or = 0.5 MAC, P < 0.05), halothane (> or = 1.0 MAC, P < 0.01) and isoflurane (> or = 2.0 MAC, P < 0.01) induced a significantly higher expression of P-selectin on the surface of platelets, indicating the degranulation of alpha-granules. In the presence of desflurane (> or = 0.5 MAC, P < 0.05), halothane (> or = 1.0 MAC, P < 0.01), isoflurane and sevoflurane (both > or = 2.0 MAC, P < 0.01), a redistribution of GPlb occurred, indicating platelet activation. N2O had no effect. In conclusion, several of the volatile anaesthetics tested in vitro induced changes in the expression of P-selectin and GPlb, being characteristic of platelet activation. None of the anaesthetics investigated interfered with the platelet response to ADP stimulation.

Characterization of the human fMLP receptor in neutrophils and in Xenopus oocytes

N‐formyl peptides (e.g. fMLP; N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine) are potent mediators for inflammatory reactions. We report functional expression in Xenopus oocytes of human fMLP‐R98 cDNA, without co‐expression of the promiscuous G‐protein subunit, Gα‐16. Stimulation of voltage‐clamped oocytes (−70 mV) with fMLP produced a dose‐dependent biphasic inward current with fast and slow components. Analysis using GTP‐γ‐S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G‐protein of the Gq family. The fast current reversed at −25 mV and was blocked by SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid), suggesting the current is carried by Cl−. The slow current showed weak inward rectification, was Ca2+‐dependent and blocked by Cd2+, 4‐AP (4‐aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels. Comparative experiments with human neutrophils using flow cytometric analysis showed that the proportion of neutrophils activated by fMLP was reduced in the presence of SITS, in the absence of external calcium and in the presence of Cd2+, TEA (tetraethylammonium) and haloperidol but not 4‐AP. In addition, the oxidative burst from activated neutrophils was reduced by SITS and by the absence of external calcium but not by Cd2+, TEA, 4‐AP or haloperidol. We suggest that in human neutrophils activation by fMLP is dependent on store‐operated calcium influx that appears to be regulated by Cl− channels and linked, in part, to non‐selective cation channels.

Thiopental impairs neutrophil oxidative response by inhibition of intracellular signalling.

BACKGROUND AND OBJECTIVE Thiopental in clinically relevant concentrations inhibits the oxidative function of neutrophils, whereas only very high, non-therapeutic concentrations of methohexital induce a similar effect. The study characterized the molecular basis of this differential action of oxy- and thiobarbiturates on neutrophils. METHODS Neutrophils were incubated in vitro with thiopental or methohexital using concentrations within the therapeutic range. Neutrophil responses were induced using different stimuli: N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), C5a and 1,2-dioctanoyl-sn-glycerol (DiC8-DAG). FMLP and C5a bind to specific G-protein-coupled receptors that share the same second messenger cascade. In contrast, DiC8-DAG, an activator of protein kinase C, bypasses the signal transduction pathway downstream of the receptors. Hydrogen peroxide production by neutrophils was assessed using flow cytometry. To characterize the localization of the interaction site, FMLP receptor expression and cytosolic-free calcium were further analysed. RESULTS FMLP and C5a-induced hydrogen peroxide production were both significantly impaired by thiopental, but not by methohexital. When postreceptor signalling was bypassed, by stimulation with DiC8-DAG, neither thiopental nor methohexital affected hydrogen peroxide production. Additionally, neither of the barbiturates impaired the cytosolic Ca2+ response. CONCLUSIONS We conclude that neither protein kinase C nor the hydrogen peroxide-generating enzymes are affected by thiopental or methohexital. The unimpaired Ca2+ response suggests that the function of the receptors and G-proteins were also unimpaired. Taken together, this indicates that the site of action of thiopental is in the cellular signalling upstream of protein kinase C.

Inhibition of the neutrophil oxidative response by propofol: preserved in vivo function despite in vitro inhibition.

Background and objective: Propofol has been shown to inhibit a variety of functions of neutrophils in vitro, but there is a lack of in vivo data. To analyse the effects of propofol on neutrophil function in vivo we chose to investigate cataract surgery since it represents a small surgical procedure with minimal immunomodulatory effects induced by surgery. We sought to analyse any immunosuppressive effects of propofol after short‐term administration in vivo in comparison to local anaesthesia as well as to in vitro effects of propofol. Methods: The study was designed as an open randomized trial enrolling 20 patients undergoing general or local anaesthesia. The neutrophil oxidative response and propofol plasma concentration were assessed prior, during and after anaesthesia. Neutrophil function was determined flow cytometrically based on dihydrorhodamine 123 oxidation. Results: Propofol concentrations which yielded a marked suppression in vitro did not alter the neutrophil oxidative response during cataract surgery in vivo. However, after local anaesthesia the neutrophil oxidative response declined to 37%, compared to the control response prior to anaesthesia. Conclusions: Although we could detect the well established suppression of neutrophil function by propofol in vitro it was not evident in vivo. This may be due to compensating effects on neutrophil function during surgery in vivo. The decline in the neutrophil oxidative response in the local anaesthesia group might be due to increased stress and catecholamine concentrations or a direct interaction of local anaesthetics with neutrophil intracellular signalling.

Less adrenergic activation during cataract surgery with total intravenous than with local anesthesia

Background: Previous studies reported that, contrary to local anesthesia, cataract surgery under inhalational anesthesia is associated with substantial adrenergic activation. We tested the hypothesis that total intravenous anesthesia (TIVA) with propofol and alfentanil produces less or comparable adrenergic activation during cataract surgery than local anesthesia.