Screening for bacteremia in sepsis and renal failure using hemofilters for renal replacement therapy.

BACKGROUND: In patients with sepsis and renal failure, extracorporeal blood flow during renal replacement therapy may lead to the deposition of bacteria on artificial membranous surfaces, which might be suitable for the detection of pathogens. We studied whether discarded dialysis hemofilters can be used for the detection of bacteremia in patients with sepsis and renal failure. METHODS: Hemofilters of 16 ICU patients with sepsis were sampled. The hemofilters were incubated with soy broth and dehisced under sterile conditions. Samples were plated on blood agar and analyzed. Patient's characteristics were assessed. RESULTS: Despite the use of antibiotics in 87.5 % (14/16), a true positive detection rate of 31.3 % (5/16) for bacteremia was found by using cultures from hemofilters. The overall true positive rate of blood cultures was significantly lower (10.7 %, 8/75, p = 0.048). Bacteria detected in hemofilters were similar to those found in blood cultures or by cultures from other sources of infection in 80 % (4/5). CONCLUSIONS: Cultures from used hemofilters of patients with sepsis and renal failure provide the opportunity to identify pathogenic microorganisms as an add-on approach. Further studies should investigate whether this method is applicable in clinical practice to enhance the sensitivity of microbiological diagnostics.

Validation of a procedure to assess ASA-response in patients with decreased, initial TRAP induced aggregation.

Whole blood aggregometry on the is a simple, fast and standardized method and it is widely used to assess platelet function under antiplatelet therapy. Reference ranges and a cut-off value as a measure of ASA response were established by measuring arachidonic acid induced aggregation (ASPI-test) in healthy volunteers and cardiac patients after and used to classify patients as ASA responders or non-responders. However, assessing the platelet function is highly affected by pre-analytical and analytical conditions and often reduced aggregation by TRAP induced aggregation (TRAP-test) is seen, rendering the samples difficult for interpretation of the ASPI-test and the responder status to ASA. We hypothesised that in this simplified model any preanalytical factor has the same effect on TRAP-testing as on ASPI-testing and that by calculating the ratio between a defined, normal TRAP-test result and the TRAP-test result measured for the individual patient this ratio could be applied to the measured ASPI-test thereby reaching a more valid discrimination between ASA responders and -non-responders. TRAP- and ASPI-test were performed in blood from ASA-treated volunteers and controls on Multiplate before an after pneumatic tube delivery as a model to stimulate shear stress induced platelet activation and aggregation. The calculated, normalised ASPI test result after tube delivery did not differ significantly from the initial ASPI test result although tube delivery had a significant impact on the measured ASPI test result. If applied to patients samples a definite judgement on the ASA response status of patients with reduced "general platelet activatability" could be given. Normalisation of the ASPI-test result using the TRAP-test result may provide a method to judge on the ASA response status in patients with decreased initial "general platelet activatability".

Factors that contribute to spontaneous platelet aggregation and streptokinase-induced aggregation in whole blood.

When whole blood is stirred there is a "spontaneous" platelet aggregation (SPA) which is presumed to be caused by proaggregatory factors released from platelets and other blood cells. Adding streptokinase (SK) to stirred whole blood frequently increases the rate and extent of the platelet aggregation that occurs; this is likely to be via immune complex formation between SK and natural anti-SK antibodies leading to increased release of pro-aggregatory factors. In this investigation we have examined the effects of several inhibitors and antagonists in an attempt to identify the proaggregatory factors that contribute to both SPA and SK-induced aggregation (SKA) and to evaluate different means of inhibiting both processes. The effects of the inhibitors/antagonists were determined in vitro after adding them to citrated whole blood obtained from healthy volunteers. Platelet aggregation was measured using a platelet counting technique. Inhibition of both SPA and SKA by apyrase and by FPL 66096 (a P2T receptor antagonist) demonstrated the involvement of ADP in both processes. Inhibition by chlorpromazine indicated that the most likely source of the ADP is red cells. The effects of sulotroban (a TXA2 antagonist) indicated involvement of TXA2 in SKA but not in SPA. The lack of effect of specific antagonists at S2, alpha 2 and PAF receptors suggested lack of involvement of serotonin, catecholamines and platelet-activating factor in either SPA or SKA. Both SPA and SKA were potently inhibited by low concentrations of iloprost (a PGI2 analogue), but a high concentration of SIN-1 (a NO donor) was much less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of arachidonic acid-dependent Ca++ influx in human platelets.

Quin2 was used to study the rise in cytoplasmic free calcium ([Ca++]i) and the role of prostaglandin (PG) endoperoxides/thromboxane A2 (TxA2), reduced glutathione (GSH), ADP and the glycoprotein (GP) IIb-IIIa complex in mediating [Ca++]i rise during arachidonic acid (AA)-induced platelet aggregation. Ca++ mobilization, mostly due to an influx across the plasma membrane, is completely inhibited by aspirin and persists after selective blockade of TxA2 synthase by dazoxiben. GSH total depletion causes a complete aggregation block and 90% inhibition of the transient: U-46619, a stable analog of cyclic endoperoxide PGH2, stimulates [Ca++]i transient in aspirin-treated or in GSH-depleted platelets. ADP-scavengers, ATP (which competes for the ADP receptor), and monoclonal antibodies against the GP IIb-IIIa complex reduce AA-induced Ca++ influx. Therefore, PG endoperoxides alone or a PGH2/TxA2 mimetic stimulate Ca++ influx. Synthesis of PGH2 and TxA2 depends on the availability of GSH, which acts as the reducing cofactor for the PG-peroxidase activity. ADP and GP IIb-IIIa are regulating factors of AA-mediated Ca++ influx during platelet activation.

The platelet glycoprotein IIb/IIIa complex is involved in the adhesion of activated platelets to leukocytes.

The adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counter-receptor (CD15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

Platelets inhibit the activity of platelet-activating factor acetylhydrolase in monocyte-derived macrophages.

Blood platelets are capable of interacting with monocytes and macrophages and of enhancing various functions of these cells, which are believed to play a role in thrombosis and inflammation. An increase in the uptake of oxidised low density lipoprotein (LDL), in the synthesis of procoagulant tissue factor, thrombospondin and leukotrienes, as well as stimulation of oxygen radical production by platelets has been described (1-5). In circulating blood, a substantial proportion of monocytes was found to be associated with platelets, but the pathophysiological significance of such platelet-monocyte conjugates is not yet clear (6,7). Immigration of monocytes into the arterial intima and their differentiation into macrophages are initial steps in the development of an atherosclerotic lesion (8). During differentiation, there is a tremendous increase in the activity and secretion of the enzyme PAF acetylhydrolase (PAF = platelet-activating factor = 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (9,10), and there is some evidence that this enzyme may contribute to the development of atherosclerosis. It cleaves PAF, and the remaining lyso-PAF is chemotactic for monocytes (11). Furthermore it also acts on oxidised low density lipoproteins and enhances their uptake into macrophages (12,13). We were therefore interested in investigating whether platelets may modulate the differentiation of monocytes into macrophages and the activity of PAF acetylhydrolase.

The role of the GSH-disulfide status in the reversible and irreversible aggregation of human platelets.

Disturbance of cellular SH/SS status of blood platelets by diminution of the level of reduced glutathione is very sensitively reflected in changes of the in vitro aggregation. Additionally, disulfide-linked protein polymers are formed. One of these polymers participates in mediating platelet disaggregation.

An extract of feverfew inhibits interactions of human platelets with collagen substrates.

The interaction of platelets with surfaces coated with collagens of type III (C III) or IV (C IV) has been studied by measuring the deposition of 51-Cr-labeled platelets and by scanning electron microscopy (SEM). Experiments were performed using platelet-rich plasma (PRP) and suspensions of gel-filtered platelets (GFP). Platelets were deposited on C III mainly as surface-bound aggregates. In contrast they were deposited on C IV mainly as spread forms of individual cells. Formation of aggregates on C III was more extensive for PRP than for GFP; in contrast platelet spreading on C IV was more extensive for GFP than for PRP. The effects of an extract of the plant feverfew on platelet-collagen interactions were determined. Feverfew extract inhibited the deposition of 51-Cr-labeled platelets on both C III and C IV in a dose-dependent way. Similar concentrations of extract were needed to inhibit the formation of surface-bound aggregates and to inhibit platelet spreading in both PRP and GFP.

Influence of diamide on aggregation, cytoskeletal proteins, and arachidonic acid metabolism in human platelets.

Simultaneous addition of diamide (azodicarboxylic acid-bis-dimethylamide, DIA), a SH-oxidizing agent, and collagen causes a deaggregation of otherwise irreversibly aggregating platelets. Thromboxane B2 (TXB2) and 12-HE-TE formation is inhibited depending on the concentration ratio between collagen and DIA. Thus, at 0.25 mM DIA and 20 micrograms/ml collagen neither TXB2 nor 12-HETE were measurable, but a full scale reversible aggregation is induced. Deaggregation is further attained by adding DIA to collagen-induced aggregates at a time, when maximum amplitude has been achieved. Investigation of arachidonic acid (AA) metabolites under these conditions revealed no influence of DIA on AA metabolism. Therefore, AA metabolization seems to play a minor role in collagen-induced aggregation and DIA-induced deaggregation. Polymerization of certain cytoskeletal proteins of the platelets, after addition of DIA, parallels DIA-induced deaggregation. DIA inhibits endogenous AA release, probably by interaction with platelet plasma membrane. DIA seems to inhibit the release of the alpha-granula protein thrombospondin.

Effect of GSH depletion by 1-chloro-2,4-dinitrobenzene on human platelet aggregation, arachidonic acid oxidative metabolism and cytoskeletal proteins.

Platelet reduced glutathione (GSH) is completely depleted by 1-chloro-2,4-dinitrobenzene (CDNB), which is a substrate for GSH-S-transferase. GSH-depleted platelets: a) aggregate normally at high inducer concentration; b) respond with increased (after arachidonic acid) or depressed (after collagen) aggregability at low inducer concentration; c) show almost no arachidonic acid-induced stimulation of the hexose monophosphate shunt; d) are sensitized to oxidant agents such as diamide, which elicits a faster cytoskeletal protein oxidative polymerization and reversible aggregation. Our results suggest that GSH acts as a reducing cofactor and/or free radical scavenger in the PG-hydroperoxidase step of the cyclooxygenase pathway; moreover, GSH protects membrane and cytoskeletal protein -SH groups from oxidation.

Functional behaviour of mononuclear blood cells from patients with hypercholesterolemia.

Mononuclear cells were prepared from venous blood obtained from 20 patients with a newly diagnosed hypercholesterolemia and without clinical signs of vascular disease, and from 19 age and sex matched controls. Adhesiveness to plastic surface, phagocytic activity measured as ingestion of zymosan particles, and spontaneous motility of mononuclear cells from patients were significantly higher by 57%, 19% and 50%, respectively, when compared to controls. In controls chemotaxis induced by the chemotactic peptide FMLP was slightly higher than spontaneous motility measured in absence of FMLP, whereas in patients FMLP significantly inhibited cell motility by about 47%. With the exception of FMLP-induced chemotaxis the results indicate that mononuclear cells are hyperreactive in hypercholesterolemia.

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages.

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.

Helenalin and 11 alpha,13-dihydrohelenalin, two constituents from Arnica montana L., inhibit human platelet function via thiol-dependent pathways.

This study investigates the effect on human platelet function of two sesquiterpene lactones from Arnica montana L., helenalin (H) and 11 alpha,13-dihydrohelenalin (DH). Both compounds inhibited collagen-induced platelet aggregation, thromboxane formation and 5-hydroxytryptamine secretion in a concentration-dependent manner at 3-300 microM. When arachidonic acid was used as stimulus, thromboxane formation remained unaffected despite of inhibition of platelet aggregation. Both H and DH reduced the number of acid-soluble sulfhydryl groups in platelets, by up to 78% at anti-aggregatory concentrations. Moreover, H- and DH-induced platelet inhibition could be prevented by the thiol containing amino acid cysteine. It is concluded that H and DH inhibit platelet function via interaction with platelet sulfhydryl groups, probably associated with reduced phospholipase A2 activity.

In vitro dose response to different GPIIb/IIIa-antagonists: inter-laboratory comparison of various platelet function tests.

AIMS: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration-response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). METHODS: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 microg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose-response curves were established on the basis of a sigmoidal Imax)-model [I=(Imax)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50) 0.84 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/ml). A similar pattern was observed for abciximab. CONCLUSIONS: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the "real" inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.

Studies on the effects of agonists and antagonists on platelet shape change and platelet aggregation in whole blood.

The shape change that occurs when platelets are stimulated with an agonist can be quantitated by monitoring changes in their forward-scatter/side-scatter profile using a flow cytometer. Here we have stimulated platelets in citrated whole blood with several agonists and determined the time-course and extent of the shape change that occurs. In some experiments parallel investigations of shape change and aggregation were performed. Aggregation was measured by monitoring the fall in number of single platelets using a Whole Blood Platelet Counter. Some agents (ADP, PAF, U46619 and 5HT) produced a strong and rapid change in platelet forward-scatter/side-scatter that was maximal within 10 s. Others (A23187 and collagen) produced a strong but slower response. Adrenaline produced only a weak response that was also slow to develop, and PMA did not produce any response. The concentrations of each of ADP, PAF, U46619 and 5HT needed to induce a shape change were lower than those required for aggregation. Selective PAF, TXA2 and 5HT antagonists (WEB 2086, sulotroban and MCI-9042) clearly inhibited both the shape change and the aggregation induced by the appropriate agonist; in each case the effect of the antagonist was to move the dose-response curve to the right. These results are consistent with the shape change and aggregation brought about by each of these agonists being mediated via a single receptor. In contrast, a selective P2T purinoceptor antagonist (ARL 66096) markedly inhibited the aggregation induced by ADP but was found to have little or no effect on shape change. This is consistent with these platelet responses to ADP being mediated by different receptors, with P2T receptors mediating only the aggregation response.

Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.

Aggregation behaviour of blood granulocytes in patients with periodontal disease.

This behaviour was studied in 17 patients with severe marginal periodontitis and in 16 healthy controls. Aggregation induced by platelet-activating factor (PAF-acether), a potent mediator of inflammation, was significantly enhanced in the patients, whereas no significant difference was observed between patients and controls when aggregation was induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP). When the patients were subdivided into categories of progressive adult periodontitis, juvenile or post-juvenile periodontitis, aggregation induced by PAF-acether was enhanced in all three subgroups. However, FMLP-induced aggregation was slightly increased only in progressive adult and post-juvenile periodontitis, but decreased in juvenile periodontitis.

Defective function of blood granulocytes in patients with diabetes mellitus-associated marginal periodontitis.

In ten periodontitis patients suffering from type I diabetes mellitus, phagocytic activity, aggregation and chemiluminescence generation of blood granulocytes were determined. Compared to controls with clinically healthy periodontal conditions, the phagocytosis of zymosan particles and the aggregation response induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine were significantly decreased, whereas aggregation induced by the platelet-activating factor, a potent mediator of inflammation, was significantly enhanced.

Ex vivo investigation of blood monocyte and platelet behaviour in pigs maintained on an atherogenic diet.

Domestic pigs aged 4 months were fed for 16 weeks an atherogenic diet rich in cholesterol and saturated fatty acid. The increase of plasma cholesterol and triacylglycerol levels was found to be accompanied by a significant increase in the number of blood monocytes and platelets when compared to control animals. Furthermore, the atherogenic diet produced a small but significant reduction in the blood monocyte phagocytic capacity and adhesion to plastic surface. No significant differences between both groups were found when spontaneous platelet aggregation in whole blood was studied. However, platelets from pigs fed the atherogenic diet had a smaller mean cell volume compared to controls. The results indicate than an atherogenic diet may affect blood monocytes and platelets in pigs.