Molecular Studies of the Protein Complexes Involving Cis-Prenyltransferase in Guayule (Parthenium argentatum), an Alternative Rubber-Producing Plant.

Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-Binding Proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here, we identified four guayule genes - three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products organize PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP-expressing yeast efficiently incorporated 14C-isopentenyl diphosphate into dehydrodolichyl diphosphates; however, NR with high molecular weight could not be synthesized in in vitro assays. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPTs, guayule transcriptomics analysis indicated that the PaCPT3 is predominantly expressed in stem and induced by cold-stress, suggesting its involvement in NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule.

A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis.

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro.

Inhibition of itch-related responses at spinal level in rats.

The goal was to develop a rat model for determination of the effects of intrathecally administered drugs on the peripherally induced pruritic behaviors. After chronic intrathecal catheterization, a serotonin derivative (5-methoxytryptamine: MeOT, 200 µg on both sides) was injected into the lower leg skin. After the first period (phase 0: 0-30 min) MeOT injection was repeated and opioid antagonist naltrexone (10 µg), NMDA receptor antagonists ketamine (10-100 µg), kynurenic acid (1-10 µg) or their combinations were injected intrathecally. The second observational period lasted for 60 min (phases I and II, 30-60 and 60-90 min, respectively). MeOT produced pruritic behavior with high degree of interindividual differences. The second MeOT injection caused an enhanced pruritic behavior in Phase I. Naltrexone decreased the pruritic activity, while neither doses of ketamine influenced the effects of MeOT. The higher doses of kynurenic acid resulted in notable decreases in the pruritic behavior. The combinations of naltrexone with ketamine or kynurenic acid produced a prolonged antipruritic effect. Our data suggest an important direction for the development of a new itch model in rats that focuses on the spinal mechanism of itching. Besides, the results revealed the role of the spinal opioid and NMDA receptors in this process.

Anticoagulant effects of an antidiabetic drug on monocytes in vitro.

INTRODUCTION: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. METHODS: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry). RESULTS: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. CONCLUSIONS: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.

Miniaturisation of a capillary electrophoresis microchip for the sensing of endocrine disruptors.

Although capillary electrophoresis amperometric detector (CE-AD) involving double-T microchannel configuration is a powerful analytical tool in terms of sensitivity and selectivity, its long microchannel configuration hinders further miniaturisation. Therefore a twisted CE microchannel configuration was used in the present study to fabricate CE-AD devices for detection of endocrine disruptors. The analyte separation time varied slightly for the twisted microchannel structure, whereas the detector sensitivities were similar for the two configurations. The conventional indium tin oxide amperometric detector in the device with twisted microchannel configuration was later modified with Prussian blue to enhance the sensitivity of detection.

Influence of jasmonic acid on endogenous gibberellin and abscisic acid in salt-stressed chard plant.

The endogenous gibberellin (GA) and abscisic acid (ABA) contents as an effect of different application times of jasmonic acid (JA) in chard seedlings exposed to salt stress were investigated. Endogenous ABA content was increased by JA treatment after NaCl treatment, rather than after JA application before NaCl treatment. JA application after NaCl treatment caused higher ABA content than treatment with 160 mM NaCl alone. Total gibberellin content decreased after NaCl stress, but NaCl-reduction in total GA contents counteracted by exogenous JA. Total endogenous GA contents were increased in JA treatment after NaCl and were highest at 24 hr of JA application before NaCl exposure. JA treatment promoted the increase of dry weight compared to chard plant exposed to 160 mM NaCl. Thus, JA presumably induces gibberellin biosynthesis showing the promotion of growth and dry weight of chard plants under salt stress.

Calcium ionophore-induced de-encryption of tissue factor in monocytes is associated with extensive cell death.

INTRODUCTION: Cell surface tissue factor (TF) is normally encrypted, but can be activated by various cellular perturbations. Exposure of TF bearing cells to calcium ionophore has been reported to increase TF activity, de-encrypt TF, by phosphatidylserine (PS)-dependent and -independent mechanisms. Our aim has been to examine at the single cell level, if increased cell surface PS coincided with increased cell surface TF antigen, and cell death (necrosis, 7-AAD-intercalation), and relate this to monocyte- and microparticle (MP)-associated procoagulant activity. MATERIALS AND METHODS: We exposed lipopolysaccharide-stimulated, human, elutriation-purified, cryopreserved TF bearing monocytes to increasing concentrations of calcium ionophore (A23187) and measured procoagulant activity in cells and supernatants. These measurements were compared with quantification of cell surface TF and PS (Annexin V) and of cell necrosis (7-AAD) by flow cytometry, and complemented by confocal microscopy. RESULTS: We observed that calcium ionophore increased cellular and MP-associated TF activity, but not cell surface TF antigen. The discrepancy between TF activity and TF antigen coincided with a dose-dependent increase in the number of cells expressing PS. These cells were to a large extent necrotic and many of them also expressed TF. CONCLUSIONS: We suggest such TF positive dying cells to contribute to the discordance between TF activity and TF expression. Calcium ionophore also increased MP-associated TF activity and release of MPs may be a way to disseminate procoagulant activity. Our findings emphasize the importance of adequately assessing cell death and taking into consideration its possible role in experiments with calcium ionophore.

Neisseria meningitidis lipopolysaccharides in human pathology.

Neisseria meningitidis causes meningitis, fulminant septicemia or mild meningococcemia attacking mainly children and young adults. Lipopolysaccharides (LPS) consist of a symmetrical hexa-acyl lipid A and a short oligosaccharide chain and are classified in 11 immunotypes. Lipid A is the primary toxic component of N. meningitidis. LPS levels in plasma and cerebrospinal fluid as determined by Limulus amebocyte lysate (LAL) assay are quantitatively closely associated with inflammatory mediators, clinical symptoms, and outcome. Patients with persistent septic shock, multiple organ failure, and severe coagulopathy reveal extraordinarily high levels of LPS in plasma. The cytokine production is compartmentalized to either the circulation or to the subarachnoid space. Mortality related to shock increases from 0% to > 80% with a 10-fold increase of plasma LPS from 10 to 100 endotoxin units/ml. Hemorrhagic skin lesions and thrombosis are caused by up-regulation of tissue factor which induces coagulation, and by inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1). Effective antibiotic treatment results in a rapid decline of plasma LPS (half-life 1-3 h) and cytokines, and reduced generation of thrombin, and PAI-1. Early antibiotic treatment is mandatory. Three intervention trials to block lipid A have not significantly reduced the mortality of meningococcal septicemia.

The synergistic antinociceptive interactions of endomorphin-1 with dexmedetomidine and/or S(+)-ketamine in rats.

UNLABELLED: Spinal administration of the endogenous mu-opioid agonist peptide, endomorphin-1, results in antinociception in rodents, but there are few data about its interaction with other antinociceptive drugs. We investigated the antinociceptive interactions at the spinal level of endomorphin-1 with the N-methyl-D-aspartate antagonist S(+)-ketamine, the alpha2-adrenoceptor agonist dexmedetomidine, or both in awake rats. Nociception was assessed by the tail-flick test. Dose-response curves were determined for endomorphin-1 (0.6-50 microg), for dexmedetomidine (0.1-10 microg), for mixtures of S(+)-ketamine (30 or 100 microg) with endomorphin-1 (2-18 microg) or of endomorphin-1 with dexmedetomidine in a fixed ratio (4:1), and for the triple combination of the three drugs after intrathecal administration. Endomorphin-1 and dexmedetomidine both produced dose-dependent antinociception. The coadministration of 100 microg S(+)-ketamine significantly enhanced the antinociceptive effect of 6 microg endomorphin-1. Isobolographic analysis of the combinations of endomorphin-1 and dexmedetomidine revealed a synergistic interaction between these drugs. The 80% effective dose for the triple combination was significantly less than that for either binary combination. These data indicate that S(+)-ketamine and dexmedetomidine, acting via different receptors, produce synergistic antinociceptive interaction with endomorphin-1 at the spinal level. Furthermore, the triple combination of an opioid agonist, an alpha2-adrenoceptor agonist, and an N-methyl-D-aspartate receptor antagonist shows potent antinociceptive activity. IMPLICATIONS: The coadministration of the N-methyl-D-aspartate antagonist receptor antagonist, S(+)-ketamine, or the specific alpha2-adrenoceptor agonist, dexmedetomidine, significantly enhances the antinociceptive effect of the endogenous mu-opioid agonist, endomorphin-1, at the spinal level. The triple combination of the three drugs causes a further improved antinociception.

Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes.

After developing and applying a method for cryopreserving monocytes, we found a substantial cell loss when culturing these cells. Monocytes were isolated from blood donors by density gradient centrifugation, purified by elutriation and cryopreserved. Thawed cells were cultured in ultra low attachment wells and studied with Annexin V, Propidium iodide, Dihexyloxacarbocyanine (DiOC(6)(3)), bromolated deoxyuridine triphosphate nucleotides (Br-dUTP), DNA ploidy and DNA ladder methodologies. The main cell loss was within the first 24 h and recovery on day 7 was 35-40%. The first 2-6 h of culture were found to be crucial for determining which cells survive. Initially (2-4 h), apoptosis was the main feature but after 6 h, necrosis dominated. Two populations of cells developed after 24 h: "A" consisting of larger cells with low levels of apoptosis and necrosis signals and population "B" comprising smaller cells with a high expression of necrotic but low levels of apoptotic signals. Signs of DNA fragmentation were slight. These early, dynamic changes may be important for the interpretation of experimental results when investigating monocytes in culture.

Non-mannose-capped lipoarabinomannan stimulates human peripheral monocytes to expression of the "early immediate genes" tissue factor and tumor necrosis factor-alpha.

In the present study, we have shown that stimulation of cryopreserved, human peripheral blood monocytes with the cell wall components from Gram-negative bacteria, lipopolysaccharide (LPS), and from rapid-growing Mycobacterium sp., non-mannose-capped lipoarabinomannan (AraLAM), both induce expression of the "early immediate genes" tissue factor (TF) and tumor necrosis factor-alpha (TNF-alpha). This was demonstrated both at the protein and the mRNA levels. Antibodies against the CD14 receptor could block the stimulating effects. AraLAM was a significantly weaker inducer than LPS, and we speculate that this may reside in the number of the fatty acids in the part of the molecule that interacts with the CD14/Toll-like receptors (TLR). Finally, both LPS and AraLAM activated the "early immediate genes" through translocation of the transcription factor proteins NF-kappaB/Rel and increasing the binding activity of AP-1.

Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience.

BACKGROUND: Monocyte purification by means of counter-current elutriation and subsequent cryopreservation for future use was initiated in 1986 and has been established as a routine since 1993. AIM: To sum up and evaluate our method for the isolation and preservation of monocytes. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor blood by density gradient centrifugation, and monocytes were isolated from the PBMC by counter-current elutriation centrifugation using the Beckman J-6M/E centrifuge. The monocytes were then cryopreserved at 135 degrees C and thawed when required for experimental use. RESULTS: Results are given for the last 6 years, including 59 elutriations and the fractions containing monocytes. The mean purity of monocytes was 93% (range 64-98%); mean recovery was 51% (range 22-55%). Studies of CD14 expression and Annexin V indicate that there are no differences between elutriated fractions immediately upon purification or after freezing and thawing. The studies also indicate that interdonor variations are much larger than intradonor variations. DISCUSSION: Although it differs from other reports in certain respects, our procedure has nevertheless produced results in line with other findings. After extensive testing and use in different contexts we feel confident that we have established a method for producing a large number of purified and well-preserved monocytes. CONCLUSION: The goal of being able to perform a large number of experiments with monocytes of high purity and good functionality has been reached.

Aspirin potentiates LPS-induced fibrin formation (FPA) and TNF-alpha-synthesis in whole blood.

The effect of aspirin on LPS-incubation of whole blood was investigated. Aspirin induced a concentration dependent increase (2.5-5-fold at 5 mM aspirin) in LPS-induced appearance of TNF-alpha and fibrinopeptide A (FPA) in plasma, despite the concomitant increase in the inhibitory cytokine IL-100. Aspirin substantially raised the levels of LPS-induced TF-mRNA and TNFalpha-mRNA in monocytes isolated from whole blood. The median ratio for TF-/beta-actin mRNA increased from 1.5 +/- 0.44 in the presence of LPS-alone, to 2.5 +/- 0.51 when 5 mM aspirin was added. The TNFalpha/beta-actin mRNA ratios were 1.8 +/- 0.4 and 5.5 +/- 2.7 respectively. Addition of exogenous PGE2 before incubation nearly abrogated the effect of aspirin on TNF-alpha, substantiating the role of PGE2 as a regulator of TNF-alpha synthesis, whereas the effect on FPA was small. Thus, in the presence of LPS in this whole blood model, aspirin apparently had a pro-inflammatory rather than an anti-inflammatory effect.

The effects of ketamine and its enantiomers on the morphine- or dexmedetomidine-induced antinociception after intrathecal administration in rats.

BACKGROUND: The spinal administration of some N-methyl-d-aspartate receptor antagonists results in antinociception and potentiates the effects of opioids and alpha2-adrenoceptor agonists, but ketamine and its enantiomers have not been examined. The present study investigated the interactions of racemic ketamine, R(-)-ketamine and S(+)-ketamine with morphine and with dexmedetomidine. METHODS: Intrathecal catheters were implanted into male Wistar rats. Three days later, the acute nociceptive sensitivity was assessed using the tail-flick test. Analgesic latencies were converted to the percentage maximum possible effect. The dose that yielded 50% of the maximum possible effect (ED50) and dose-response and time-course curves were determined for the ketamines (30-300 microg), morphine (0.1-3.0 microg), dexmedetomidine (0.3-10.0 microg), and mixtures of two doses of ketamines (30 or 100 microg) with different doses of morphine or dexmedetomidine for fixed-dose analysis. RESULTS: Neither racemic ketamine nor its enantiomers alone had a significant effect on the tail-flick test, with the exception of the highest dose of racemic ketamine, which caused motor impairment. Morphine and dexmedetomidine each produced dose-dependent antinociception, with ED50 of 1.7 microg (95% confidence interval: 1.04-2.32) and 4. 85 microg (3.96-5.79), respectively. A low dose (30 microg) of racemic ketamine or its enantiomers did not influence the ED50 of morphine significantly. Coadministration of 100 microg racemic ketamine or S(+)-ketamine, but not R(-)-ketamine, significantly enhanced and prolonged the antinociceptive effect of morphine. Both doses of racemic ketamine or its isomers significantly decreased the ED50 value for dexmedetomidine, although the higher dose of racemic or S(+)-ketamine had the highest potency. One-hundred micrograms of racemic ketamine or S(+)-ketamine also prolonged the effects of dexmedetomidine. CONCLUSIONS: These data indicate that racemic ketamine and S(+)-ketamine, but not R(-)-ketamine, exhibit similar effectiveness in potentiating the antinociceptive effects of both morphine and dexmedetomidine.

Cellular activating properties and morphology of membrane-bound and purified meningococcal lipopolysaccharide.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

Macrophage tropism: fact or fiction?

Primary HIV-1 isolates can be distinguished by phenotypic qualities such as the ability to productively infect cells of established CD4-positive lines and to induce syncytia in MT-2 cells. Such viral phenotypes have also been reported to confer host cell specificity. It is perceived that primary isolates with the syncytium-inducing phenotype (SI or rapid/high) are T cell tropic and are therefore unable to infect primary cells of the monocyte/macrophage lineage. However, we have consistently found that these isolates are as capable of establishing infection in monocyte-derived macrophages (MDM) as the monocytotropic, non-syncytium-inducing variants (NSI or slow/low). It is known that differentiation, activation, and proliferation of human monocytes are affected by both isolation methods and culture conditions. Therefore, to test whether our inability to discriminate macrophage tropic HIV-1 isolates could be explained by differences in culturing techniques, we isolated monocytes by elutriation or short-term adherence and allowed the cells to mature and differentiate in either the presence or absence of autologous lymphocytes. After removal of nonadherent cells, MDM were infected with a panel of SI and NSI primary HIV-1 isolates. MDM were susceptible to infection by the SI as well as the NSI isolates, regardless of whether or not the cells were allowed to mature in the presence of autologous lymphocytes. However, MDM matured in the presence of autologous lymphocytes replicated HIV-1 isolates (both NSI and SI) to a higher titre than MDM matured in the absence of lymphocytes. In light of these findings and recently published reports on HIV-1 phenotype and chemokine receptor usage we believe that the term macrophage-tropic strains of HIV-1 is no longer appropriate.

Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alfa (TNF-alpha) in human monocytes.

We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (TNF-alpha), as well as the prostaglandin PGE2 in human monocytes. Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level, and reduced PGE2 production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs.

Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13.

Exposure of monocytes to pro-inflammatory cytokines or lipopolysaccharide (LPS) may induce synthesis and expression of tissue factor (TF). In this paper we have focused on the induction of TF-activity in human monocytes by the pro-inflammatory cytokines recombinant human interleukin 1 (rhIL-1 alpha) (rhIL-1 beta) (rhIL-6) and human tumour necrosis factor alpha (rhTNF-alpha), measured as procoagulant activity (PCA) in a microtitre plate-based clot assay. In addition we have studied the modulation of IL-1 alpha/beta induced TF-mRNA and PCA by rhIL-4, rhIL-10 and rhIL13. IL-1 alpha and IL-1 beta induced a concentration dependent increase in TF-activity. Neither IL-6 nor TNF-alpha gave rise to procoagulant activity at the concentrations tested (0.2-20 ng/ml). IL-4, IL-10 and IL-13, all effectively diminished IL-1 alpha/beta induced PCA, shown at the protein- and at the mRNA-level, while cell viability was unaffected. These results add to the previously demonstrated role of IL-4 and IL-10 as inhibitors of LPS-induced TF-activity, showing that these anti-inflammatory cytokines are not specific for LPS-activation but interfere with other stimulating substances such as IL-1, which may be involved in diseases where LPS is not present.

Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes.

We have developed a functional assay to study the inflammatory capacity of plasma collected from patients with severe gram-negative septic shock. In this assay, elutriation-purified, cryo-preserved human monocytes from one healthy donor are combined with plasma from patients with severe persistent septic shock for 5 h. Subsequently, the plasma is removed, medium added, and procoagulant activity (PCA) and secretion of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) measured after 18-h incubation. Plasma from 10 patients (6 died) infected with Neisseria meningitidis previously shown to contain high levels of native lipopolysaccharide (LPS) (median 2,700 pg/ml), TNF-alpha, IL-6, IL-8, and complement activation products, had a low net spontaneous inflammatory capacity on the monocytes. The median levels of PCA, TNF-alpha, and IL-6 were 5, 0, and 4%, respectively, of the monocyte activities induced by normal plasma boosted with purified N. meningitidis (Nm)-LPS (2,500 pg/ml; net LPS-boosted capacity, 100%). The levels of PCA, TNF-alpha, and IL-6 obtained with plasma from shock patients were not different from those induced by plasma from 10 meningococcal patients without shock or with plasma from healthy persons. Boosting shock plasma with 2,500 pg/ml Nm-LPS had little effect on the monocyte activities since the median values of PCA, TNF-alpha, and IL-6 revealed a minimal increase from 5, 0, and 4% to 9, 2, and 6%, respectively. The shock plasmas revealed a strong LPS-inhibitory capacity that was largely absent in plasmas from 10 meningococcal patients without shock since the median levels of PCA, TNF-alpha, and IL-6 increased from 5, 0, and 0% to 135, 51, and 73%, respectively, after boosting with 2,500 pg/ml Nm-LPS. The LPS-inhibitory capacity was closely associated with the levels of IL-10. The median levels of IL-10 were 19,000 pg/ml in nine shock patients vs. 22 pg/ml in nine nonshock patients with systemic meningococcal disease. Removal of native IL-10 by immunoprecipitation restored the capacity of plasmas to induce monocyte activation either by native LPS or by boosting with Nm-LPS. IL-4 and TGF-beta were not detected in shock plasmas. In 24 patients with detectable meningococcal LPS ( > 10 pg/ml, 0.1 endotoxin units/ml), the levels of IL-10 were correlated to the levels of LPS (r = 0.79, P < 0.001). IL-10 declined from initiation of antibiotic therapy and paralleled the levels of native LPS. Decreasing levels of IL-10 in serially collected shock plasmas were directly related to increasing monocyte responsiveness after Nm-LPS boosting. These results suggest that IL-10 plays a major role in containing activation of monocytes and possibly other LPS-responsive cells during overwhelming meningococcemia.

LPS-induced release of EGF, GM-CSF, GRO alpha, LIF, MIP-1 alpha and PDGF-AB in PBMC from persons with high or low levels of HDL lipoprotein.

We have examined basal and lipopolysaccharide (LPS)-induced release of epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated peptide alpha (GRO alpha), leukaemia inhibitory factor (LIF), macrophage inflammatory protein-1a (MIP-1 alpha) and platelet-derived growth factor-AB (PDGF-AB) in peripheral blood mononuclear cells (PBMC) from 20 persons with either high (n = 10) or low (n = 10) levels of high-density lipoprotein (HDL). PBMC were incubated with 100 ng LPS/ml for up to 160 h, and showed a significantly higher release of the chemokines GRO alpha (P = 0.04) and MIP-1 alpha (P < 0.01) in persons with high HDL, whereas levels of GM-CSF were similar. Levels of EGF, LIF and PDGF-AB were always low, and remained unaltered during 160 h of incubation. These findings indicate that PBMC from persons with high or low levels of HDL have different functional properties, of importance in cell recruitment and activation.