Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Evidence for the involvement of carbohydrate moieties in the adhesion of U937 cells to tumor necrosis factor-α (TNFα)-stimulated vascular endotheliumin vitro

Treatment of U937 cells with fructose 1-phosphate (P) and fucoidan dose-dependently inhibited the adhesion of these monocytic cells to TNF-stimulated human umbilical vein eindothelial cells (HUVEC) (IC₅₀=1 mM and 10 g/ml respectively). These carbohydrates (CHO) failed to inhibit U937 adhesion to unstimulated (basal) HUVEC or phorbol 12, 13 dibutyrate (PdBu)-stimulated HUVEC. At 10 mM concentration, both fucose 1-P and lactose 1-P inhibited TNF-stimulated adhesion while the latter also inhibited basal adhesion. Fructose 6-P, fucose, galactose 1-P, glucose 1-P, glucose 6-P, glucuronic acid,-glycerol 1-P, mannose 1-P, mannose 6-P, ribose 1-P and ribose 5-P tested at 10 mM did not inhibit U937 cells adhesion to basal or TNF-stimulated HUVEC. These data suggest that CHO may play an important role in modulating monocytes adhesion to cytokine-induced adhesion molecule(s) on the surface of HUVEC.     

Exogenous Nitric Oxide Increases Neutrophil Adhesion to Cultured Human Endothelial Monolayers through a Protein Kinase G Dependent Mechanism

Endothelial-neutrophil adhesion is a critical step in acute inflammatory diseases, which is mediated in part by P-selectin and platelet-activating factor (PAF). Nitric oxide (NO) is well known as an endogenous second messenger derived from endothelial cells, and regulates many important physiological events, however, the direct effects of NO on endothelial-neutrophil adhesion is less well understood. The objective of this study was to examine whether, and how relatively high levels of exogenous NO increases neutrophil adhesion with respect to P-selectin and PAF. Endothelial monolayers were exposed to chemical agents for 30 min, and the adhesion of ^5lCr-labeled neutrophils measured in a static adhesion assay. Spermine-NONOate (SNO), an NO donor, significantly increased neutrophil adhesion and expression of P-selectin at a concentration of 1 mM. SNO (1 mM)-mediated neutrophil adhesion was significantly inhibited by a protein kinase G inhibitor, KT5823 (0.5 M), but not by a classical protein kinase C inhibitor, Gö6976 (10 nM), a tyrosine kinase inhibitor, genistein (1 M), or a protein kinase A inhibitor, H-89 (0.1 M). P-selectin surface expression induced by 1 mM SNO was also significantly inhibited by 0.5 M KT5823. Conversely, a cytoplasm calcium chelator, TMB-8 (0.1 mM), significantly exacerbated both the neutrophil adhesion and P-selectin expression induced by SNO. WEB 2086 (10 M), a PAF receptor antagonist, blocked neutrophil adhesion, but did not block P-selectin expression induced by SNO. These data suggest that NO increases endothelial-neutrophil adhesion through protein kinase G-mediated P-selectin mobilization to the cell surface and endothelial PAF synthesis.     

Benoxaprofen inhibits the adhesion of human monocytes to cultured vascular endothelium

Pretreatment of human monocytes with benoxaprofen for at least 2 h produced a dose-dependent abrogation of their adhesion to monolayers of cultured porcine endothelium with 0.05 g/ml and 50.0 g/ml of the drug inducing a mean 33% and 83% inhibition of adhesion respectively. When the endothelium was treated with the drug there was no modification of monocyte adhesion. In contrast, pretreatment of endothelium with 5.0 and 50.0 g/ml benoxaprofen for at least 6 h, resulted in a mean 35% and 31% inhibition of polymorphonuclear cell (PMN) adhesion in 6/11 experiments. This inhibitory effect was not seen when drug-treated PMNs were added to endothelium. An impairment of monocyte chemotactic migration was only apparent with high concentrations of the drug (50 g/ml). These results suggest that an important anti-inflammatory property of benoxaprofen is the inhibition of monocyte adhesion to vascular endothelium.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Effect of recombinant human interleukin 2 on neutrophil adherence to endothelial cells in vitro

Human neutrophils were demonstrated to possess interleukin-2 receptor (IL-2R) and chains, not chain and the binding of IL-2 to the IL-2R chain on neutrophils plays an important regulatory role in neutrophil functions. We have investigated in this study the hypothesis that recombinant human IL-2(rhIL-2) can directly activate human neutrophils and increase their adherence to human umbilical vein endothelial cells (HUVEC). In an in vitro microtiter adherence assay, rhIL-2 significantly stimulated neutrophil adherence to HUVEC in a dose- and time-dependent manner. rhILI-2 concentration at 2000 u/ml and 2 hour incubation gave the best neutrophil stimulation. Treatment of neutrophils with rhIL-2 increased the expression of adhesion molecule CD18. Pretreatment of the stimulated neutrophils with a blocking monoclonal antibody to CD18 decreased but not completely blocked the adherence of neutrophils to HUVEC. These data suggest that rhIL-2 can directly stimulate and increase neutrophil adherence to HUVEC by enhancing the expression of CD18 and possibly other adhesion molecules on neutrophil surface. This may be a critical step in the early stage of the vascular leak syndrome (VLS) associated with high dose IL-2 therapy.     

Soluble β2-μ-Associated and β2-μ-Free HLA Class I Heavy Chain Serum Levels in Interferon-α Nonresponder Chronic Hepatitis C Patients. Markers of Immune Activation, and Response to Antiviral Retreatment

The serum levels of soluble ₂--associated and ₂--free HLA class I heavy chains were determined in 28 interferon- nonresponder chronic hepatitis C patients retreated with interferon- plus ribavirin and in 70 healthy subjects. The baseline levels of ₂--associated and ₂--free HLA class I heavy chains were significantly higher in patients than in healthy controls(P = 0.001). The levels of ₂--associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment(P = 0.03). At the sixth month of treatment and after 6 months of follow up the levels of ₂--associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of ₂--free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble ₂--associated HLA class I heavy chains, as a marker of immune activation, could identify interferon- non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon- plus ribavirin.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Effects of neuroblastoma tumor gangliosides on platelet adhesion to collagen

Gangliosides, sialic acid-containing glycosphingolipids, enhance tumor formation in experimental animals and are associated with tumor progression and metastasis in humans. The mechanism(s) for this activity is (are) unknown. One possibility is enhanced platelet activation, since the interaction of platelets with tumor cells contributes to tumor cell arrest in the vascular compartment. We have previously shown that neuroblastoma tumor gangliosides (NBTG) enhance platelet adenosine triphosphate (ATP) secretion, aggregation, and adhesion. We determined that these NBTG effects are specific for collagen and are mediated through an (( integrin-dependent mechanism. This report describes the effects of NBTG on a physiologically relevant model of collagen-( interaction. Platelet adhesion to immobilized native collagen fibers similar to those found in the extracellular matrix of blood vessels was determined. Platelet adhesion is enhanced by NBTG in a concentration-dependent manner. Incubation with concentrations of 1 and 10 m NBTG increased platelet adhesion by 9% and 52%, respectively, compared to less than 1% in controls not incubated with gangliosides (P = 0.001 and P < 0.0001, respectively). In addition to increasing the number of adherent platelets, NBTG promoted more rapid attachment. In NBTG-incubated platelets, platelet adhesion began after a 5-min lag phase and was maximal at 30 min compared to a 20-min lag phase and maximal adhesion at 60 min for control platelets. At 30 min this difference was significant (P = 0.017); however, by 120 min there was no difference between NBTG and controls (P = 0.259). NBTG also induces platelet adhesion at collagen concentrations (0.1 g) that failed to support adhesion of control platelets. These effects of NBTG require Mg or Mn ions but are not supported by Zn or Ca ions. Furthermore, preincubation of platelets with a blocking antibody (6F1) to the integrin collagen receptor ( abrogates all of the effects of NBTG. These results indicate that tumor gangliosides enhance platelet adhesion to extracellular matrix collagen and promote rapid stabilization of the collagen-(( interaction, the initial steps in platelet activation.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Bradykinin Antagonizes the Effects of α-Thrombin

-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1M AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 × 10^–6 cm/sec to 4.92 × 10^–6 cm/sec. BK (1M) produced a similar increase to 4.46 × 10^–6 cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1M), prior to addition of AT, reduced the AT permeability coefficient to 2.69 × 10^–6 cm/sec. Addition of NA-BK (1 M) for 5 minutes, then BK (1 M) for 5 minutes, inhibited the effect of BK and of AT (1 M) on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 × 10^–6 cm/sec). AT (1 M) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 M) for 2 minutes also inhibited the effects of -thrombin (1 M) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an -thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 M) with -thrombin (1 M) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators -thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.     

Susceptibility to beta-lactam antibiotics in septicemia isolates from twenty-nine European laboratories

In 1984 the European Study Group on Antibiotic Resistance (ESGAR) consecutively collected gram-negative bacilli and staphylococci blood isolates and performed susceptibility testing with 11 antibiotics using the microdilution method. In all 2,578 isolates were collected: 68% gram-negative bacilli and 32% staphylococci. The MICs of ampicillin and cefazoline for the susceptible gram-negative bacilli were 1–8g/ml; of piperacillin0.5–4; of Sch 34343, cefotaxime, moxalactam, ceftazidime and aztreonam0.5–2g/ml; of cefoxitin, cefuroxime and cefamandole0.5–8g/ml. For susceptible staphylococci the MICs of cefazoline and cefuroxime were0.5–1g/ml, and of cefoxitin, moxalactam, ceftazidime and cefotaxime,0.5–32 g/ml. The resistance levels varied between laboratories and countries, being lower in Northern Europe. In clinical protocols on patients with gram-negative septicemia from whom cefazoline-resistant strains were isolated, cefotaxime was the beta-lactam most commonly used (12%). In protocols on patients with staphylococcal septicemia from whom gentamicin-resistant or cefazoline-resistant strains were isolated, the most commonly used beta-lactam was cloxacillin (6%).     

Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells

Mechanisms underlying facilitation by dopamine of extracellular adenosine 5-triphosphate (ATP)-activated current were investigated in rat pheochromocytoma PC12 cells using the whole-cell voltage-clamp techniques. Dopamine (10 and 100 M) augmented the peak amplitude of an inward current elicited by ATP (3–100 M). The activation time course of the ATP-evoked current was accelerated by dopamine; the presence of 10 M dopamine shifted the dependence of activation rate constants on the concentration of ATP toward a lower concentration range two fold. Dopamine also accelerated the inactivation and the deactivation, which was determined from the current decay upon washout of ATP. Intracellular mediators responsible for the dopamine-induced facilitation was estimated by loading various compounds in patch pipettes. Facilitation was not observed when K-252a (1 M), a protein kinase inhibitor, was included in the intracellular solution. In addition, facilitation was also attenuated by intracellular adenosine 5-O-(thiotriphosphate)tetralithium salt (ATPS (1 mM) or --methylene ATP (1 mM). Inclusion of adenosine 3, 5-cyclic monophosphate sodium salt (cAMP, 100 M), guanosine 3,5-cyclic monophosphate sodium salt (cGMP, 100 M), 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 M) or phorbol-12,13-dibutylate (1 M) in the intracellular solution did not affect the facilitation. Guanonsine 5-O-(thiotriphosphate)tetralithium salt (GTPS, 500 M) or guanosine 5-O(2-thiodiphosphate)-trilithium salt (GDPS, 500 M) did not modify the facilitation either. The results suggest that dopamine augments the ATP-activated inward current by facilitating association of ATP to its binding site, and that the augmentation may be mediated through some protein kinase which is different from cyclic-nucleotide-dependent protein kinases or protein kinase C.     

NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-B in the presence of CM by studying the degradation rate of individual IB isoforms.Results: We show that, as expected, CM induced NF-B translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF- and IL1 naturally present in the CM. However, both the amplitude of NF-B translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF- and IL-1, either separately or together. Our results show that CM induced a faster degradation of the IB- and IB- isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-B activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF- mediated endothelial activation.Received 5 October 2003; returned for revision 2 December 2003; accepted by A. Falus 19 May 2004     

Expression of immunoglobulin genes in common variable immunodeficiency

Five common variable immunodeficiency (CVI) patients were analyzed for expression of immunoglobulin (Ig) genes. In the pokeweed mitogen (PWM)-induced Ig-production assay, the combination of T and B cells showed that all patients' T cells had normal helper functions and all patients' B cells had profound defects. The defective B-cell maturation stages based on their Ig gene expression patterns were variable. One of five patients showed normal -chain gene expression and nearly normal IgM production, but neither IgG nor IgA production, which suggested that this patient's B-cell defects might lie on a - to or - to class-switch stage. B cells in another patient showed low -chain gene expression and low IgM production, but an Ig enhancer region, which is an important region for expression of Ig genes, was intact. Thus, this patient might have a transacting factor defect which interacts with the Ig enhancer region. The other three patients showed no -chain gene expression and no IgM production. Thus, their B-cell defects lay on the B-cell maturation stage, similar to X-linked agammaglobulinemia. These results showed that primary B-cell defects in CVI occurred at several B-cell differentiation stages, which could be recognized by expression of Ig genes.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

Relation between cell na extrusion and transtubular absorption in the perfused toad kidney: the effect of K,ouabain and ethacrynic acid

Summary Transtubular absorption of Na and Cl, and intracellular ion concentrations were evaluated in toad kidneys perfused with solutions containing K and without K, and in the presence of 1 mM Ouabain and 1 mM Ethacrynic acid. The following values were obtained with 8.5 mM K: Transtubular absorption of Na and Cl68% (percent of filtered load); cell content 294 mole Na, 433 mole K, 100 mole Cl/g solids. Lack of K in the perfusate diminished transtubular absorption to 25% and the cells gain 244 mole Na/g solids, and lose an equimolecular quantity of K. The process is reversible upon raising the K concentration in the perfusate. Ouabain inhibits transtubular absorption to 6%; the cells lose about 110 mole K/g solids, but cellular Na is maintained at the control levels. Ethacrynic acid inhibits transtubular absorption to 3%; the cells approximately double their Na and Cl content, but their K is maintained at the control levels. These observations cannot be explained exclusively in terms of an effect on the distal tubule. Probably proximal as well as distal tubules are involved. A single Na pump seems insufficient to account for all experimental findings. The existence of two separate pumps is therefore proposed.     

Analysis of mutations introduced into the chromosomal immunoglobulin μ gene

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin gene. Homologous recombination between the transferred mutant C region and the wild-type chromosomal C region is expected to introduce the 2-bp deletion into the chromosomal gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal gene deletions.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Antibacterial cathelicidin peptide CAP11 inhibits the lipopolysaccharide (LPS)-induced suppression of neutrophil apoptosis by blocking the binding of LPS to target cells

Objective: The action of antibacterial cathelicidin CAP11 (cationic antibacterial polypeptide of 11 kDa) on the lipopolysaccharide (LPS)-induced suppression of neutrophil apoptosis was evaluated in vitro.Methods: Human neutrophils (10⁶ cells/ml) were incubated alone or with mononuclear cells (6 × 10⁵ cells/ml) in the presence of LPS (10 ng/ml) and CAP11 (0.1 ~ 10 g/ml), and neutrophil apoptosis was determined.Results: LPS suppressed neutrophil apoptosis, accompanied with the activation of NF-B, phosphorylation of extracellular signal-related protein kinase (ERK), expression of Bcl-XL (an anti-apoptotic protein) and inhibition of caspase 3 activity. Interestingly, CAP11 (>1 g/ml) reversed the actions of LPS to trigger these changes, and induced neutrophil apoptosis (p < 0.0001). Moreover, neutralizing antibodies against Mac-1 (CD11b/CD18) and Toll-like receptor (TLR) 4 completely blocked the LPS-induced suppression of neutrophil apoptosis (p < 0.0001), suggesting a major role of Mac-1 and TLR4 in the LPS-mediated neutrophil activation. In addition, LPS activated monocytes to produce proinflammatory cytokines (IL-1, TNF- and IL-8) and inhibited neutrophil apoptosis. Importantly, CAP11 (>1 g/ml) reduced the cytokine production, thereby inducing neutrophil apoptosis (p < 0.0001). Finally, CAP11 (>1 g/ml) strongly suppressed the LPS-binding to neutrophils and monocytes (p < 0.01).Conclusions: CAP11 is able to block the LPS-induced survival of neutrophils via the suppression of anti-apoptotic signaling in neutrophils and cytokine production from monocytes by inhibiting the binding of LPS to target cells.Received 21 April 2004; returned for revision 10 June 2004; accepted by M. Katori 14 June 2004