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.

[Differential diagnosis of platelet disorders]. / Differenzialdiagnose von Thrombozytenstörungen.

Platelet disorders frequently represent a cause of bleeding disorders with a late manifestation and spontaneous bleeding. Disturbances of cellular hemostasis can be of quantitative nature due to an altered production or destruction of platelets. Qualitative disturbances can be associated with defects of adhesion, secretion or degranulation. Drug induced reactions, inflammatory processes and autoimmune reactions are the most frequent underlying disorders. Even a late manifestation, however, does not exclude congenital disorders. In the differential diagnosis of thrombocyte disorders the anamnestic analysis of the clinical circumstances of manifestation, of a family background and potentially interfering drugs are of central importance. Template bleeding time, aggregometry and flow cytometry are complementary methods for the characterization of functional defects. First of all, a von Willebrand syndrome as the most frequent congenital form of a mucocutaneous bleeding pattern needs to be excluded. The clinical context is very important in the analysis of disturbances of platelet turnover. Reticulated platelets allow the quantitative assessment of reduced production or increased destruction. Platelet indices, morphological assessment of blood and bone marrow and immunological tests allow the pathogenetical classification of thrombocytopenia. Idiopathic thrombocytopenia (ITP) is a frequent diagnosis by exclusion. The analysis of glycoprotein expression and the genetic characterization of suspected congenital defects are only performed in selected cases. Clinical and laboratory assessment are complementary in the discrimination of secondary forms of thrombosis from clinically relevant clonal disturbances.

Monitoring of peripheral blood cytotoxic T-cells under fluvastatin treatment in renal transplant recipients.

Flow cytometric T-cell analysis is capable of adding valuable information for balancing immunosuppression in transplant recipients as it can take into account individual effects of immunosuppressive drugs on each patient as well as effects of other drugs which may modify the overall immunosuppression. Studies suggest that HMG-CoA-reductase-inhibitors (statins) reduce the frequency of organ rejection, although the precise mechanism of this effect is unknown. We therefore evaluated the effect of fluvastatin on size and activation of T-cell subpopulations and NK-cell activity in renal transplant recipients. At baseline, the population size of activated (HLA-DR+) T-cells was negatively correlated to serum HDL cholesterol suggesting an increased T-cell activation at low HDL levels. Fluvastatin treatment of a hypercholesterolemic group of patients for two months significantly decreased the LDL cholesterol. A longitudinal analysis revealed a relative increase in non-MHC restricted cytotoxic T-cells (CD3+/CD16+ or CD56+) over time which was significantly attenuated in fluvastatin treated patients but not in normocholesterolemic controls. Moreover, a relative decrease of activated MHC class I-restricted cytotoxic CD8+ T-cells was only observed upon fluvastatin treatment. NK-cell number and activity did not differ between groups. In summary, fluvastatin treatment of hypercholesterolemic renal transplant recipients is associated with a specific modulation of T-cells exerting cytotoxic effector functions.

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

BACKGROUND: 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. METHODS: Blood was taken from healthy donors. Neutrophils were pretreated with cytokines (interleukin [IL]-1beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNFalpha]; 0.01-10 ng/ml) and stimulated with fMLP (100 nM) in vitro. Functional and phenotypical parameters were quantified flow cytometrically. RESULTS: The oxidative burst response increased after priming with 0.1 ng/ml TNFalpha, 1 ng/ml GM-CSF, or 10 ng/ml IL-8. Upregulation of fMLP receptors, CD11b, and CD66b and downregulation of CD62L showed a close correlation to the oxidative burst response. Altered expression of these parameters partly reached significance at lower cytokine concentrations in comparison with the oxidative burst. IL-1beta and IL-6 had no effect. CONCLUSIONS: Our results showed that the expression of phenotypical parameters closely correlates with functional parameters in human neutrophils. Thus an up- or downregulation of antigens such as CD11b or CD62L reflects cytokine-induced functional changes.

Blood leukocyte subsets and cytokine profile after autologous peripheral blood stem cell transplantation.

High-dose chemotherapy with autologous peripheral blood stem cell transplantation (PBSCT) includes the risk of infectious complications due to neutropenia and therapy-induced immune deviation. In order to understand early immune recovery in this situation, we analyzed the distribution of cell subsets by flow cytometry and we measured cytokine production in a whole blood assay stimulated with lipopolysaccharide (LPS) in order to induce monocyte (MO) activation in 43 patients with solid tumors or lymphoma treated with two cycles of high-dose chemotherapy and PBSCT. Blood was collected at the following time points: before start of mobilization chemotherapy, before and after high-dose chemotherapy, and 10 and 30 days after PBSCT. In the lymphocyte compartment, we found a depletion of B cells and naive T cells and a transitory reduction of natural killer (NK) cells, whereas MO and neutrophils recovered rapidly. However, during early recovery, HLA-DR expression on MO and the percentage of CD16(+) MO was considerably reduced. Production of proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-alpha upon LPS stimulation was severely impaired directly after chemotherapy and unexpectedly remained low during early recovery of myeloid cells, whereas production of IL-1RA was enhanced, indicating a shift of immune competent cells to an anti-inflammatory or anergic state early after PBSCT.

Autologous tandem transplantation: almost complete reduction of neutropenic fever following the second transplantation by ex vivo expanded autologous myeloid postprogenitor cells.

Reduced post-transplant performance status because of infectious complications is still a problem following autologous peripheral blood stem cell transplantation (aPBSCT). In this study, a tandem transplantation scheme for 15 patients with breast cancer including etoposide (1500 mg/m(2)), ifosfamide (12 g/m(2)) and carboplatin (1500 mg/m(2)) as conditioning regimens, followed by aPBSCT, was used to evaluate the potential clinical benefit of the additional retransfusion of low numbers of ex vivo expanded committed myeloid postprogenitor cells (PPCs) (median 408 x 103 CFU-c/kg BW, range 0.93-1995) following the second transplantation. Following a 7+2 days expansion (using recombinant human SCF, IL-1beta, IL-3, IL-6 + G-CSF), CFU-c generated from CD34-positive cells from leukapheresis products could be expanded by a median factor of 153 (range 5-434). Flow cytometric analysis and morphology of CFUs have shown a nearly exclusive expansion and differentiation of committed myeloid progenitor cells and a significant reduction of CD34-positive cells. In an intra- and interindividual comparison it could be shown that the retransfusion of committed myeloid postprogenitor cells significantly accelerates myeloid recovery. Although retransfusion of PPCs fails to abrogate severe neutropenia following aPBSCT, it significantly ameliorates infectious complications and shortens the duration of hospital stay.

Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens.

BACKGROUND: Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis. METHODS: After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested. RESULTS: The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum. Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions. DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide. Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells. CONCLUSIONS: These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies.

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.

Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.

The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.

Increased histidine decarboxylase expression during in vitro monocyte maturation; a possible role of endogenously synthesised histamine in monocyte/macrophage differentiation.

OBJECTIVE: In this study the expression of histidine decarboxylase (HDC), the pivotal enzyme in histamine formation and the effect of endogenously produced histamine on differentiation antigens was examined during in vitro differentiation of human monocytes. MATERIAL AND TREATMENT: Human elutriated monocytes from healthy volunteers were incubated with macrophage colony stimulating factor (M-CSF) and the expression of HDC was followed at both mRNA and protein levels. To study the possible function of histamine we followed the expression of some cell surface markers (CD14, CD16, CD91, CD49d and CD11c) relevant for phagocytic differentiation upon incubation in the presence of different histamine inhibitors, an HDC inhibitor: S(+)-alpha-fluoromethyl-histidine HCl, (alphaFMH), a compound that disturbs the interaction of histamine with intracellular cyp450 moieties: N,N-diethyl-2-[4-(phenylmethyl) phenoxy]-ethanamine HCI, (DPPE); and H1 and H2 receptor antagonists, Triprolidine and Cimetidine. RESULTS: During in vitro culture of elutriated human monocytes, in the presence of M-CSF, the gene expression and biosynthesis of HDC was considerably increased. The various antihistamine agents decreased the expression of the cell surface markers examined in this study. CONCLUSIONS: These data support the elevation of HDC expression during human monocytic differentiation and the possibility that monocyte-derived histamine is partially involved in regulation of M-CSF induced in vitro human monocyte/macrophage phagocytic differentiation.

The influence of the duration of cardiopulmonary bypass on coagulation, fibrinolysis and platelet function.

BACKGROUND: The duration of cardiopulmonary bypass (CPB) might influence blood coagulation. This appears particularly relevant in the light of new, less invasive techniques that propose smaller incisions at the expense of a possible prolongation of time on CPB. METHODS: The time-dependent effects on coagulation, fibrinolysis and platelet function were investigated in 94 patients scheduled for elective coronary artery bypass grafting. Tests on coagulation, fibrinolysis, and platelet function (flow cytometric assay of expression densities of glycoprotein IIb/IIIa and P-selection were performed the day before surgery and after completion of surgery. RESULTS: A significant correlation was found between the duration of CPB and parameters of increased coagulation, decrease of platelet counts during CPB and platelet function. Longer duration of CPB led to an increased need for transfusion of red blood cells. CONCLUSIONS: The duration of CPB affects thrombin formation as well as platelet count and function, but not the fibrinolytic system. This may prove to be a disadvantage when employing minimally invasive techniques that prolong the duration of CPB.

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages.

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.

Enzymatically degraded LDL preferentially binds to CD14(high) CD16(+) monocytes and induces foam cell formation mediated only in part by the class B scavenger-receptor CD36.

Heterogeneity of peripheral blood monocytes is characterized by specific patterns in the membrane expression of Fc gamma-receptor III (FcgammaRIII/CD16) and the lipopolysaccharide receptor (LPS receptor CD14), allowing discrimination of distinct subpopulations. The aim was to analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with modified lipoproteins by the use of either enzymatically degraded low density lipoprotein (E-LDL), acetylated low density lipoprotein (ac-LDL), oxidized low density lipoprotein (ox-LDL), or native low density lipoprotein. Highest E-LDL binding was observed on CD14(high) CD16(+) monocytes as determined by flow cytometry, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly isolated peripheral blood monocytes. This was accompanied by upregulation of the 2 class B scavenger receptors CLA-1/SR-BI (CD36 and LIMPII Analogous-1/scavenger receptor type B class I) and CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger-receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. In predifferentiated monocyte-derived macrophages, an anti-CD36 antibody inhibited cellular binding and uptake of E-LDL by approximately 20%, suggesting that recognition of these hydrolase-modified low density lipoprotein particles is mediated only in part by the class B scavenger receptor CD36.

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

OBJECTIVES: To determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 --> Glu; PstI 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. DESIGN: Case control study of patients with sepsis. SETTING: Intensive care units within university hospitals. PATIENTS: A total of 204 patients diagnosed with sepsis and 250 healthy blood donors. INTERVENTIONS: None. 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. CONCLUSIONS: Our 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.

Calystegines in root cultures of Atropa belladonna respond to sucrose, not to elicitation.

Calystegines are norpseudotropine alkaloids accumulating in root cultures of Atropa belladonna, together with tropine derivatives, e.g. hyoscyamine. Both alkaloid groups are derived from the tropane alkaloid pathway. For the investigation of the regulation and individual steps of tropane biosynthesis, methods for the induction of the pathway were tested. Elicitation by chitosan, or defence responses to ABA and methyl jasmonate did not enhance calystegine accumulation, but led to a more or less pronounced decrease. By blocking one arm of the diverged tropane pathway, calystegine accumulation can be increased, but total tropane alkaloid formation does not increase considerably. By elevation of sucrose supply, both, total alkaloids and calystegines in particular were increased approximately threefold. The mechanism of the induction of the biosynthesis by sucrose is not known and needs further experiments.

Inhibition of effects of endogenously synthesized histamine disturbs in vitro human dendritic cell differentiation.

Histamine, a principal mediator in various physiological and pathological cell functions is synthesized from L-histidine exclusively by histidine decarboxylase, an enzyme, which is expressed in many tissues of mammalian organism. Histamine plays a role in various cellular functions, including cell differentiation. The aim of this study was to determine the presence and to characterize the role of the endogenously produced histamine during in vitro dendritic cell (DC) differentiation induced by interleukin-4 (IL-4) and granulocyte-monocyte colony stimulating factor (GM-CSF). The changes in intracellular histamine content, biosynthesis and gene expression of histidine decarboxylase were investigated during this process. One also studied how histamine receptor antagonists and a histamine synthesis blocker influence the expression of differentiation antigens on the DC during in vitro maturation. During in vitro differentiation parallel culture incubations were performed by adding H1 receptor antagonist triprolidine, H2 receptor antagonist tiotidine, the tamoxifene derivate DPPE which blocks the intracellular binding of histamine, and an irreversible blocker of histidine decarboxylase, alpha-fluoromethyl histamine (alpha-FMH). The results show simultaneous increase in both histidine decarboxylase level and histamine content during differentiation of elutriated monocytes toward DC. Both blockade of de novo histamine production (by alpha-FMH) and inhibition of histamine binding (by H1 and H2 receptor antagonists, triprolidine and tiotidine, respectively) markedly decreased CD40 expression and that of CD45 from the 3rd day of treatment. DPPE by disturbing intracellular interaction of histamine with cytochrome P-450 moieties was able to decrease the expression of CD45, CD86, HLA-DR, CD33, CD40 and CD11c. Based on the data it is suggested that endogenous histamine is actively synthesized during cytokine-induced in vitro DC differentiation. The functional relevance and autocrine and paracrine action of endogenously produced histamine is supported by the data showing that inhibition of histamine synthesis by HDC, blocking of histamine binding by both 'extracellular' histamine receptors (by specific antagonists, triprolidine and tiotidine) and 'intracellular' antagonists (DPPE) disturb the differentiation of DC. This conclusion is supported by the fact, that by the inhibition of histamine acting in an autocrine/paracrine way, the expression pattern of differentiation markers on DC is markedly changed.

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response.

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP. In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.