Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.     

Ethanol induced changes in superoxide anion and nitric oxide in cultured microglia

Induction of oxidative stress has been implicated as a causative factor in fetal alcohol syndrome although the source of reactive oxygen species is not clear. One potential source is the microglia, the CNS macrophage, which generate superoxide anion as part of their normal immune function. Our data indicate that chronic exposure to ethanol alters the function of cultured neonatal hamster microglia by inducing superoxide anion production in resting (nonstimulated) cells. An increase in superoxide anion was seen at 24 or 48 hr of ethanol treatment but was not seen during acute exposures of up to 3 hr. This effect was dose dependent and was maximal at 20 mM ethanol. Treatment with ethanol did not directly activate the respiratory burst seen in microglia and did not act as a priming agent to enhance phorbol-ester-stimulated superoxide anion production. Lipopolysaccharide-mediated priming of microglial superoxide anion production was also not affected by exposure to 20 mM ethanol for 24 hr. Ethanol treatment, however, did depress nitric oxide (NO) levels in hamster microglia which had been stimulated to produce NO by polyinosinic:polycytidylic acid (poly I:C). Uptake of latex beads was increased by 24 hr of exposure to ethanol. The overall action of ethanol on neonatal hamster microglia is to shift the balance between the production of superoxide anion and NO. Because NO is protective to mammalian cells, these changes predict that oxidative stress in the CNS would be enhanced.     

Resonant energy transfer from proteins to pyridine nucleotides in mitochondria

BACKGROUND: Anaesthetic agents inhibit certain functions of human neutrophils. The respiratory burst (RB) enzyme in the plasma membrane of neutrophils leads to the production of superoxide anion. The oxygen radicals are responsible for killing phagocytised micro-organisms. We investigated the in vitro influence of remifentanil, fentanyl, and alfentanil on the respiratory burst of human neutrophils. METHODS: For the flow-cytometric evaluation, leukocytes were obtained as supernatant following sedimentation and were incubated with the tested drugs. The concentrations in vitro were adjusted to conform to the plasma concentrations reported for anaesthesia and also to 10-fold higher concentrations. The RB was measured by intracellular oxidation of dihydrorhodamine to fluorescent rhodamine after induction of phorbol-myristate-acetate (PMA), Escherichia coli (E. coli) or priming by tumour necrosis factor alpha followed by stimulation of n-formyl-methionyl-leucyl-phenylalanine (TNF-alpha/FMLP). In order to exclude prestimulation of the neutrophil granulocytes, negative controls were carried out. Propidium iodide (PI) was added for viability discrimination immediately prior to flow cytometry measurement. RESULTS: Regardless of the triggering agents chosen (PMA, E. coli, TNF-alpha/FMLP), remifentanil, fentanyl, and alfentanil had no significant effect on the neutrophils' respiratory burst even in concentrations which were higher than those encountered during in vivo conditions. CONCLUSION: With respect to peri- and postoperative risk of infection, anaesthetics and analgetics with no inhibiting effect on neutrophil function should be used. These results show that remifentanil, fentanyl, and alfentanil do not influence the neutrophils' respiratory burst in vitro.     

Lipopolysaccharide primes human alveolar macrophages for enhanced release of superoxide anion and leukotriene B4: self-limitations of the priming response with protein synthesis

Human alveolar macrophages (AM) can produce potent reactive oxygen intermediates (ROI) and arachidonic acid metabolites (eicosanoids), which have important roles in host defense and the pathogenesis of some diseases of the lung. Bacterial lipopolysaccharide (LPS) is believed to cause profound lung injury and can prime mouse peritoneal macrophages for the enhanced secretion of ROI and eicosanoids. Therefore, we investigated the effect of LPS pretreatment on the ability of AM to release superoxide anions (O2-) and leukotriene B4 (LTB4). LPS can prime AM for the enhanced secretion of O2- and LTB4, regardless of whether they are derived from nonsmokers or smokers. Moreover, judging from the time-response characteristics, this priming for LTB4 release could be inhibited in the later stages of pretreatment, when the O2(-)-releasing capacity was enhanced. The priming inhibition was prevented, at least in part, by cycloheximide, but not by SOD and/or catalase. In addition, cycloheximide also inhibited the priming for O2- release. Hence, protein synthesis might be necessary for the priming for O2- release and for inhibiting the priming for LTB4 release. This phenomenon of self-limiting the priming response with LPS seems to be very important when we consider the high oxygen tension in the lungs and the many bacterial substances inspired into alveoli.     

Outcome of Burkholderia (Pseudomonas) cepacia colonisation in children with cystic fibrosis following a hospital outbreak

BACKGROUND: While there are reports on the outcome in adults and teenagers with cystic fibrosis of colonisation with Burkholderia (Pseudomonas) cepacia, there is little information in children. METHODS: In December 1991 only one of 115 children with cystic fibrosis attending a paediatric centre was colonised with B cepacia. Over the next 12 months there was a rapid increase with 23 (20%) becoming colonised; eighteen (79%) of these became colonised in hospital at a time that overlapped with the admission of a B cepacia positive child. Three different bacteriocin types were isolated, with one type (S22/PO) being present in 17 (74%) patients. The outcome for children who became colonised with B cepacia was compared with that in 33 children who continued to be colonised with Pseudomonas aeruginosa alone. RESULTS: Children colonised with B cepacia were older and more poorly nourished than those colonised with P aeruginosa, but did not have poorer pulmonary function. After colonisation, the forced expiratory volume in one second (FEV1) deteriorated between consecutive annual tests, with the average deterioration being greater in those with higher initial levels. Five children with B cepacia died from respiratory failure although none showed a fulminant deterioration. Introduction of segregation measures within hospital led to a dramatic decrease in the number of newly colonised patients. CONCLUSIONS: This study provides further evidence for person-to-person spread of B cepacia and confirms the effectiveness of simple isolation measures in interrupting spread. Colonisation with B cepacia and P aeruginosa in children is associated with a more rapid decline in lung function and a significantly increased mortality compared with cases colonised with P aeruginosa alone.     

Cefepime versus imipenem-cilastatin as empirical monotherapy in 400 febrile patients with short duration neutropenia. CEMIC (Study Group of Infectious Diseases in Cancer)

Interleukin-8 (IL-8) priming was studied in neutrophils to examine its dependency on altered calcium fluxes and for similarity to lipopolysaccharide (LPS). IL-8 caused a rapid rise in [Ca2+]i that returned to baseline values by 20 min. Peak [Ca2+]i transients in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) were unaltered in IL-8-primed compared with unprimed cells. In comparison to LPS and tumor necrosis factor (TNF), IL-8 was a much weaker priming agent as measured by either O2- or H2O2 production. Despite their large disparity in potency, IL-8 and LPS printing were additive using fMLP, a receptor-dependent stimulator, and synergistic using the post-receptor, protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) to trigger the respiratory burst. In contrast, IL-8 and TNF priming were synergistic for fMLP (P = 0.05), but completely nonadditive when PMA was used as the neutrophil stimulant (P = 0.05 for subadditivity). Thus, lasting alterations in [Ca2+]i are not a necessary characteristic of IL-8-primed cells. IL-8 and LPS appear to prime by non-overlapping pathways, whereas IL-8 and TNF appear to share mechanisms distal to protein kinase C activation. IL-8 and LPS may independently contribute to neutrophil-mediated host defense or injury by priming through distinct pathways.     

Glycosylation improves the priming effect exerted by recombinant human granulocyte colony-stimulating factor (lenograstim) on human neutrophil superoxide production

The role of glycosylation in modulating the activity of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) on polymorphonuclear leukocytes (PMNs) was investigated. We addressed this study by comparing the effects of lenograstim (glycosylated rHuG-CSF) and its deglycosylated counterpart on superoxide production by PMNs on fibronectin. When the triggering activity of the cytokine was evaluated, no O2- release was elicited from neutrophils treated with either preparation of rHuG-CSF. Instead, a clear potentiation of both fMLP- and TNF-induced respiratory burst was produced by preincubating the cells with rHuG-CSF. Such effect was found to be significantly increased when glycosylated versus deglycosylated preparation was used, leading to the conclusion that the sugar moiety of the molecule could be of importance in improving the priming activity exerted by rHuG-CSF on PMN metabolic response.     

Differential superoxide anion generation by equine eosinophils and neutrophils

Equine eosinophils and neutrophils are believed to play an important part in the protection of horses against parasitic and bacterial invasion. Eosinophils may also play a key role in the pathogenesis of equine inflammatory conditions such as the allergic skin disease, insect hypersensitivity. The factors which stimulate the respiratory burst of equine eosinophils and neutrophils are poorly understood. The first aim of the present study was to determine the effects of the phorbol ester, phorbol myristate acetate (PMA), which is believed to activate intracellular protein kinase C, and opsonised particles of serum-treated zymosan (STZ), on the production of superoxide anions by equine eosinophils and neutrophils. Since histamine has been detected after antigen challenge in the skin of horses with insect hypersensitivity, the second aim was to establish the effects of this mediator on superoxide anion production by equine eosinophils and the receptor sub-type(s) that mediate histamine-induced responses. For comparison, responses of neutrophils from the same horses were also examined. PMA and STZ induced significant increases in superoxide anion generation by equine eosinophils and neutrophils. The estimated maximum (EMAX) superoxide anion production by eosinophils in the presence of PMA was significantly greater than that of neutrophils; the estimated concentration of PMA inducing 50% of the maximum response (EC50) by eosinophils was significantly less. The EMAX values for superoxide anion production by neutrophils in the presence of STZ were significantly greater than those for eosinophils. Histamine induced superoxide anion generation by equine eosinophils which was inhibited by the histamine-1 receptor antagonists chlorpheniramine and mepyramine, but not the histamine-2 and histamine-3 receptor antagonists, cimetidine and thioperamide, respectively. Histamine did not cause superoxide anion production by equine neutrophils. These studies demonstrate that equine granulocytes vary in their ability to produce a respiratory burst in the presence of different stimuli, with eosinophils being more responsive to protein kinase C activators and neutrophils to opsonised particles. They also show that histamine selectively induced the generation of superoxide anions by equine eosinophils via histamine-1 receptor activation. Thus, in horses with insect hypersensitivity, histamine released from cutaneous mast cells after antigen challenge could activate eosinophils which have migrated into the dermis.     

Effect of plasma and LPS on respiratory burst of neutrophils in septic patients

OBJECTIVE: To compare the respiratory burst of neutrophils in sepsis and control patients using lipopolysaccharide (LPS), autologous plasma, and a combination of the two. DESIGN: Prospective, consecutive case study. SETTING: A 16-bed intensive care unit (ICU) in a university teaching hospital. INTERVENTIONS: None. PATIENTS: Plasma was obtained from 23 healthy patients scheduled for minor surgery immediately prior to induction of anesthesia (controls) and from 23 ICU patients within 24 h of diagnosis of sepsis or septic shock. MEASUREMENTS AND MAIN RESULTS: Respiratory burst was determined by lucigenin chemiluminescence expressed as mean +/- SEM of peak values of relative light units per neutrophil. There were no significant differences between neutrophils of septic patients and controls for the stimuli saline, phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine, and LPS alone. Septic patients showed a lower respiratory burst than controls (p < 0.05) under the following stimuli: plasma alone (5911 +/- 803 vs 15,397 +/- 3038) and LPS and plasma combined (13,857 +/- 1537 vs 23,026 +/- 2640). However, when stimulated with plasma after priming with LPS, septic patients elicited a higher value than control subjects (11,373 +/- 1758 vs 5987 +/- 1234, p < 0.05). CONCLUSIONS: (1) Some components of the plasma of septic patients may have a profound effect on neutrophil response; (2) plasma as a respiratory burst stimulus differentiates between sepsis and non-sepsis samples better than other common stimuli; (3) precautions must be taken when using plasma together with LPS because of the different response depending on whether LPS-priming precedes the plasma stimulus or both are introduced simultaneously and whether septic or nonseptic plasma is used.     

The effects of panresistant bacteria in cystic fibrosis patients on lung transplant outcome

The number of cystic fibrosis (CF) patients undergoing lung transplant continues to rise as long term survival improves. One major contraindication to this potentially life-saving intervention is infection with multi-drug-resistant bacteria. We undertook this retrospective study in 66 transplanted patients over 6 yr to determine the influence of panresistant bacteria on transplant outcome. The in vitro antibiotic susceptibility pattern of respiratory tract bacteria obtained pre- and/or intraoperatively was used to categorize patients into panresistant (n = 27) (Burkholderia cepacia, n = 6, and Pseudomonas aeruginosa, n = 21) or sensitive (n = 39) groups. Postoperative ventilator days, hospital length of stay, and antibiotic days were similar for both groups (p > 0.2). The incidence of bacterial bronchitis (28% and 33%, respectively) and pneumonia (28% and 38%, respectively) did not differ between these groups (p > 0.2) at 6 mo. Likewise, one-year (81% and 83%, respectively) survival was similar for both groups (p > 0.2). As expected, panresistant B. cepacia patients had a lower 1-yr survival (50% versus 90%, p < 0.05) and had a higher mortality attributable to B. cepacia (50% versus 0%, p < 0.01) compared with panresistant P. aeruginosa patients. Our results indicate that CF patients infected with panresistant P. aeruginosa have similar transplant outcomes as patients with sensitive bacteria and should not be excluded from lung transplant based solely on this criterion.     

Interleukin-8 and GRO alpha prime human neutrophils for superoxide anion production and induce up-regulation of N-formyl peptide receptors

Interleukin-8 (IL-8) and GRO alpha are leukocyte-attracting peptides of the chemokine family. To study the priming potential of these chemokines, we measured superoxide anion production and up-regulation of N-formyl peptide receptors in human neutrophils. IL-8 and GRO alpha themselves did not stimulate production of significant amounts of superoxide anions but potentiated N-formyl peptide-induced superoxide anion production in a concentration-dependent manner. Binding measurements by flow cytometry at 37 degrees C with fluorescein-labeled N-formyl peptide revealed enhanced total N-formyl peptide binding after pretreatment of neutrophils with IL-8 and GRO alpha. Binding measurements performed at 4 degrees C indicated that the chemokines stimulated the up-regulation of N-formyl peptide receptors at the cell surface but did not alter their affinity for the ligand. This study indicates that IL-8 and GRO alpha, in addition to their known chemotactic activity, prime neutrophils for superoxide anion production, presumably by up-regulating the number of receptors for strong superoxide-anion-triggering stimuli.     

Apoptotic DNA fragmentation and upregulation of Bax induced by transient ischemia of the rat retina

We have previously shown that methionine-enkephalin (MENK) alters in dose-dependent fashion the capacity of human neutrophils to produce superoxide anion. The response of neutrophils from different donors was diverse and this effect could be due to variable activity of proteolytic enzymes involved in the degradation of the neuropeptide. In this study, we have demonstrated a highly individual aminopeptidase N (APN) activity of neutrophils from different donors. Preincubation of neutrophils with MENK, but not with the synthetic agonist of the mu (DAGO) or the delta (DPDPE) opioid receptor, down-regulated the APN activity. This was paralleled by a loss in cell surface expression of APN at physiological (10(-10) M) concentrations of MENK. The level of APN activity from different donors correlated with the effect of MENK on superoxide anion release. Neutrophils with low APN activity, if preincubated with MENK, released reduced amounts of superoxide anion. In contrast, neutrophils with high APN activity released increased amounts of superoxide anion after preincubation with MENK. Thus, the highly individual APN activity on the surface of neutrophils from different donors seems to be altered by MENK and to be related to the respiratory burst.     

Extensive aberrant Mongolian spot

Virtually all patients with cystic fibrosis (CF) die of respiratory failure resulting from chronic progressive pulmonary infections. Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia are the two major bacterial pathogens responsible for the pulmonary deterioration in these patients. Tobramycin has variable inhibitory effects on these organisms, but in many isolates this inhibition is increased in vitro by exposure to the diuretic, amiloride. Aerosolized amiloride has been shown to be of clinical benefit in CF. The basis for this synergy is unknown. To examine the possibility that amiloride-tobramycin synergy is mediated through a bacterial efflux mechanism mediated by a multidrug resistant (mdr) protein, we studied the inhibitory effect of verapamil, a known mdr inhibitor on the tobramycin MIC in P. aeruginosa and P. cepacia using standard MIC and synergy testing. Verapamil had no effect on the tobramycin MIC in P. aeruginosa but was able to act synergistically with tobramycin in reducing the tobramycin MIC markedly in all isolates of P. cepacia tested. This combination of drugs may be worth studying as a new treatment strategy for resistant P. cepacia infections.     

Regulation of renal EGF receptor expression is normal in Denys-Drash syndrome

We evaluated 819 isolates referred to us as "Burkholderia cepacia" from cystic fibrosis (CF) clinics and research laboratories from five countries; 28 (3.4%) were not B. cepacia. A further 12 (1.5%) organisms appeared to be other Burkholderia species, but identification could not be confirmed by conventional means. The most prevalently misidentified organisms were Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Comamonas acidovorans. Many of these organisms grew on oxidation-fermentation polymyxin-bacitracin-lactose (OFPBL) and Pseudomonas cepacia agars, selective media currently used for B. cepacia isolation. We developed a new medium, B. cepacia selective agar (BCSA), which is more enriched for the growth of B. cepacia yet which is more selective against other organisms than currently available selective agars. A total of 190 of 191 (99.5%) isolates of B. cepacia from patients with CF grew on BCSA without vancomycin, whereas 100% grew on OFPBL agar and 179 (94.2%) grew on P. cepacia agar. Of 189 other gram-negative and gram-positive organisms tested, 10 (5.3%) grew on BCSA without vancomycin. The addition of vancomycin to BCSA lowered the false positivity rate to 3.7% without further inhibition of B. cepacia. The false positivity rates for OFPBL and P. cepacia agars were 19.6 and 13.8%, respectively. Isolates of B. cepacia from CF patients grew most quickly on BCSA, with 201 of 205 (98.0%) being readily visible within 24 h, whereas 182 (88.8%) grew on OFPBL agar and 162 (79.0%) grew on P. cepacia agar within 24 h. We propose that the use of BCSA will allow investigators to overcome many of the difficulties associated with the identification of B. cepacia and should be considered for use as a primary isolation agar for specimens from patients with CF.     

Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure

We previously demonstrated the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of uremic patients receiving maintenance dialysis. The present study in a cohort of 162 uremic patients showed that plasma concentrations of AOPP increased with progression of chronic renal failure and were closely related to advanced glycation end products (AGE)-pentosidine (r = 0.52, p < 0.001), taken as a marker of AGE. In vivo, the relevance of AOPP and AGE-pentosidine in monocyte-mediated inflammatory syndrome associated with uremia was evidenced by close correlations between AOPP or AGE-pentosidine and monocyte activation markers, including neopterin, IL-1R antagonist, TNF-alpha, and TNF soluble receptors (TNF-sR55 and TNF-sR75). To determine the mechanisms by which AOPP and AGE could be directly involved in monocyte activation, AOPP-human serum albumin (HSA) and AGE-HSA were produced in vitro by treating HSA with oxidants or glucose, respectively. Spectroscopic analysis confirmed that AOPP-HSA contains carbonyls and dityrosine. Both AOPP-HSA and AGE-HSA, but not purified dityrosine, were capable of triggering the oxidative burst of human monocytes in cultures. The AOPP-HSA-induced respiratory burst was dependent on the chlorinated nature of the oxidant and on the molar ratio HSA/HOCI. Collectively, these data first demonstrate that AOPP act as a mediator of oxidative stress and monocyte respiratory burst, which points to monocytes as both target and actor in the immune dysregulation associated with chronic uremia.     

Interleukin-8 (IL-8), melanoma growth-stimulatory activity, and neutrophil-activating peptide selectively mediate priming of the neutrophil NADPH oxidase through the type A or type B IL-8 receptor

The capacity of neutrophils to generate superoxide (O-2) can be enhanced by prior exposure to "priming" agents such as interleukin-8 (IL-8), melanoma growth-stimulatory activity (MGSA), and neutrophil-activating peptide (ENA-78). The biological effects of these chemokines are mediated by at least two distinct receptors: type A (IL-8-RA) and type B (IL-8-RB). Using neutralizing monoclonal antibodies to IL-8-RA and IL-8-RB, we have investigated the contribution each receptor makes to the priming response. Preincubation with IL-8, MGSA, or ENA-78 enhanced the ability of neutrophils to generate O-2 following stimulation with the bacterial peptide formyl-Met-Leu-Phe. The priming effect of IL-8 was eliminated by an anti-IL-8 monoclonal antibody (mAb) that is known to bind IL-8 with high affinity and prevent receptor occupancy. Incubation of neutrophils with a neutralizing mAb specific for IL-8-RA blocked IL-8-induced priming but had no effect on priming by MGSA or ENA-78. In contrast, treatment with a neutralizing mAb specific for IL-8-RB failed to inhibit the priming effect of IL-8 but blocked both MGSA and ENA-78-induced priming. These observations indicate that the priming effect of IL-8 on the neutrophil respiratory burst is predominantly mediated via IL-8-RA, whereas priming by MGSA and ENA-78 is mediated by IL-8-RB.     

Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.     

The pharmacokinetics and protein-binding of fusidic acid in patients with severe renal failure requiring either haemodialysis or continuous ambulatory peritoneal dialysis

Antibiotic treatment options for Burkholderia cepacia infection are limited because of high intrinsic resistance. The problem is complicated by development of cross-resistance between antibiotics of different classes. We isolated antibiotic-resistant mutants by stepwise exposure to chloramphenicol (Chlor) and to trimethoprim/sulphamethoxazole (T/S) for four B. cepacia strains: ATCC13945, Per (clinical isolate), Cas and D4 (environmental isolates). Chlor(r) mutants did not produce chloramphenicol acetyl-transferase. Cross-resistance, defined as greater than four-fold increase in MIC by microtitre dilution method, was consistently seen in both types of mutants. For chloramphenicol-resistant (Chlor[r]) and trimethoprim/sulphamethoxazole-resistant (Tr/Sr) mutants of B. cepacia ATCC13945 and Cas, no MIC change was seen for piperacillin, ceftazidime, rifampicin, gentamicin, tobramycin, polymyxin B or azithromycin. B. cepacia-Per and -D4 mutants showed cross-resistance to ceftazidime and to piperacillin. Comparison of outer membrane protein (OMP) profiles of B. cepacia and their mutants by SDS-PAGE revealed Tr/Sr) mutants to be deficient in a major OMP (molecular weight 39-47 kDa). Tr/Sr mutants also expressed additional OMPs not found in wild type strains at 75-77 kDa for B. cepacia-ATCC13945 and -Cas, and 20-21 kDa in B. cepacia-D4 and -Per. No OMP changes occurred in Chlor(r) mutants. Lipopolysaccharide (LPS) profiles of each type of mutant showed new high and low molecular weight LPS bands. Cross-resistance seems to be mediated by alterations in porin and LPS for Tr/Sr mutants, but only by LPS in Chlor(r) mutants.     

Sclerotherapy for malignant pleural effusions: a prospective randomized trial of bleomycin vs doxycycline with small-bore catheter drainage

OBJECTIVES: The study's aims were to investigate the levels of gravidin, an endogenous phospholipase A2 inhibitor, in pregnancy and pre-eclampsia and to establish its effects on neutrophil function. STUDY DESIGN: Serum samples were collected from 9 nonpregnant, 15 preeclamptic, and 10 healthy pregnant women and assayed for free gravidin by enzyme-linked immunosorbent assay. Neutrophil phospholipase A2 and respiratory burst activities were determined in the presence of isolated free gravidin by cellular arachidonic acid release and superoxide anion production. RESULTS: Levels of free gravidin were higher in the healthy pregnant (36.1 +/- 5.5 ng/mL) and preeclamptic (17.8 +/- 2.8 ng/mL) groups than in the nonpregnant control group (3.9 +/- 0.5 ng/mL) and were significantly different between pregnancy groups (P <.01, Mann-Whitney U test). Free gravidin caused a concentration dependent decrease in N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil arachidonic acid release (inhibitory concentration of 50% 25 nmol/L) and superoxide anion generation (inhibitory concentration of 50% 32 nmol/L). CONCLUSIONS: Circulating levels of free gravidin are reduced in pre-eclampsia compared with normal pregnancy. This may encourage an increase in the respiratory burst of neutrophils in pre-eclampsia and could contribute to the oxidative stress and vascular damage that characterize this disease.