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PubMed1993

Simplified construction of a subtracted cDNA library using asymmetric PCR.

A novel method for the direct construction of subtracted plasmid cDNA libraries in the plasmid pBluescript is presented. Two libraries in lambda-ZAP were compared starting with general phagemid excision from both libraries. Thereafter, single-stranded (ss) plasmids from one library were subtracted with biotinylated cDNA molecules generated by asymmetric PCR on ss plasmid templates from the other library. The nonsubtracted plasmids were used to transform Escherichia coli directly, thus making a subtracted plasmid library. Preliminary data suggest that the specificity of the method is around 25%. The method is sensitive enough to detect low-abundance mRNAs. In contrast to other subtractive methods based on lambda-ZAP, the bias introduced using PCR in this case only affects the method's specificity and not its sensitivity.

Animals↗
PubMed2004

Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells.

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-alpha (TNF-alpha) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-alpha gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-zeta, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca(2+) rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-alpha gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

Cells, Cultured↗
PubMed1987

Actions of non-steroidal anti-inflammatory drugs on equine leucocyte movement in vitro.

The direct effects of four non-steroidal anti-inflammatory drugs (NSAIDs) on equine polymorphonuclear (PMN) and mononuclear (MN) leucocyte movement were investigated using two in vitro assay systems. The Boyden chamber microfilter technique measures both chemokinetic and chemotactic locomotion, and the agarose microdroplet assay measures solely chemokinesis. Zymosan-activated plasma (ZAP) and the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as standard chemoattractants for PMN and MN leucocytes, respectively. The actions of six concentrations of each NSAID, indomethacin (50 microM-10 mM), phenylbutazone (10 microM-1 mM), oxyphenbutazone (2.5 microM-500 microM) and flunixin (0.1 microM-50 microM), in suppressing cell movement induced by ZAP and FMLP were investigated. All four drugs exerted inhibitory effects on induced movement of both cell types in the Boyden chamber assay, usually in a concentration-dependent manner, although oxyphenbutazone action on PMN cells occurred only at the highest concentration tested. Significant inhibition of PMN and MN cell locomotion was produced by indomethacin, flunixin and oxyphenbutazone, and inhibition of PMN movement by phenylbutazone occurred in the agarose microdroplet assay. Flunixin was the most potent of the four drugs investigated in both assay systems. The findings may be of importance to the use of phenylbutazone and flunixin as NSAIDs in equine medicine, since the concentrations used were similar to concentrations of both drugs and the phenylbutazone metabolite oxyphenbutazone previously reported to occur in equine plasma and inflammatory exudate.

Animals↗
PubMed1987

Effects of zymosan-activated plasma and phorbol myristate acetate on isolated, perfused rabbit lungs.

The effects of complement activation on pulmonary vascular permeability are disputed. In rabbit lungs perfused with autologous blood, zymosan activated plasma (ZAP) induced a moderate increase in pulmonary vascular resistance (PVR), but did not detectably change the vascular permeability within 2 h. The stronger neutrophil granulocyte (PMN) activator, phorbol myristate acetate (PMA), usually gave larger PVR increases and also increased pulmonary vascular permeability. Lungs from neutropenic animals, similarly perfused and given PMA, showed unchanged PVR reactions but had no apparent increase in vascular permeability. Lungs perfused with cell-free medium and given PMA displayed modest PVR increases, and no measurable permeability change. The lung preparatory procedure itself markedly influenced leukocyte circulation. Exsanguination of lung donors decreased the concentration of circulating PMN significantly, and they virtually disappeared from the perfusate within minutes after start of lung perfusion. PMN-mediated effects must therefore have been caused by cells already sequestered in the lungs. We conclude that ZAP does not induce an increased pulmonary vascular permeability in isolated, perfused rabbit lungs, in contrast to PMA. The permeability effects of PMA appear to be PMN dependent.

Animals↗
PubMed1993

Effects of high dose E. coli lipopolysaccharide on rabbit lung function, and of subsequent addition of chemotactic granulocyte activators to their isolated, blood-perfused lungs.

E. coli LPS was infused (1 microgram/kg to 5 mg/kg over 30 min) to spontaneously breathing rabbits, and their arterial blood pressure (ABP), blood leukocyte count and blood gases were observed for 2.5-3.5 h. Pulmonary vascular and airway function were subsequently evaluated in vitro by comparing weight changes, fluid filtration rates, pulmonary vascular resistance (PVR) and airway pressures in their isolated, blood-perfused lungs with those in lungs from untreated rabbits. Lung preparations from both groups of animals were then exposed to autologous zymosan-activated plasma (ZAP) or n-formyl-methionyl-leucyl-phenylalanine (FMLP) and perfused for 2 more hours. LPS addition to isolated rabbit leukocytes increased cell aggregation; cell chemiluminescence after activation with FMLP was also enhanced. Infusion of 1-5 mg/kg LPS decreased the count of all types of leukocytes and caused a metabolic acidosis (BE < -8 mmol), but no decrease in ABP. PAO2-Pao2 increased by about 2.0 kPa. No vascular permeability increase was detected in the lungs of these animals during subsequent in vitro perfusion. Addition of ZAP or FMLP during perfusion markedly increased the PVR in lungs from LPS animals, but did not induce major microvascular leakage. No significant differences in edema between lungs from LPS-treated and control animals were found by microscopy.

Animals↗
PubMed2005

Brivudin compared with famciclovir in the treatment of herpes zoster: effects in acute disease and chronic pain in immunocompetent patients. A randomized, double-blind, multinational study.

OBJECTIVE: This was a double-blind, randomized multicentre trial comparing efficacy and safety of brivudin (125 mg, once a day) and famciclovir (250 mg, three times a day), both given orally for 7 days, in the treatment of herpes zoster. METHODS: A total of 2027 immunocompetent zoster patients>or=50 years with zoster-related pain at presentation were included. Outcome measures embraced prevalence of postherpetic neuralgia (PHN), defined as at least moderate pain 3 months after treatment initiation, duration of PHN, prevalence and duration of zoster-associated pain (ZAP), duration of vesicle formation and rash healing. RESULTS: The prevalence of PHN at month 3 was 11.3% with brivudin and 9.6% with famciclovir [per-protocol (PP) population]. Equivalence of the two drugs could be demonstrated (P=0.01, PP and intention-to-treat analysis). The median duration of PHN was 46.5 days with brivudin and 58 days with famciclovir (P=0.54, PP analysis). Prevalence and duration of ZAP did not differ significantly between treatment groups. The prevalence of PHN was higher in patients>or=65 years (brivudin: 16.4%, famciclovir: 16.4%), and in patients with severe rash (brivudin: 13.4%, famciclovir: 15.7%), without significant differences between treatment groups. In patients>or=65 years, median duration of PHN was shorter with brivudin than with famciclovir (39.5 vs. 57.5 days), although the difference was not statistically significant. The two drugs had equivalent efficacy in being able to accelerate the stop of vesicle formation, and lesion healing. Adverse events were similar in nature and prevalence among groups. CONCLUSIONS: The study demonstrated equivalent efficacy of brivudin and famciclovir in the treatment of herpes zoster regarding the prevention of chronic pain and the resolution of signs and symptoms of acute herpes zoster. Compared with famciclovir, brivudin provides equivalent efficacy and safety at a more convenient once-daily dose schedule.

2-Aminopurine↗
PubMed1989

Characteristics of oedema formation induced by N-formyl-methionyl-leucyl-phenylalanine in rabbit skin.

1. The characteristics of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced oedema formation were investigated in vivo in rabbit skin. 2. FMLP injected intradermally alone induced a small increase in plasma leakage, but marked synergism with prostaglandin E2 (PGE2) in producing oedema responses was observed. In the presence of PGE2, FMLP was equiactive with C5a des Arg and 100-1000 times more active than histamine in terms of permeability-increasing activity. The response to FMLP was not dependent on endogenous histamine release. 3. FMLP-induced responses were of long duration (t1/2 approximately 40-50 min) when compared with bradykinin (t1/2 approximately 4-5 min). 4. The activity of a range of N-formyl peptides in increasing vascular permeability in skin correlated well with their activity as neutrophil stimulants in vitro. 5. Intravenous infusion of zymosan-activated plasma (ZAP) resulted in transient neutropenia and inhibition of oedema formation induced by FMLP and C5a des Arg in the skin. Responses to bradykinin were unaffected by the infusion of ZAP. 6. Intravenous injection of the non-steroidal antiinflammatory drug, ibuprofen, resulted in an inhibition of FMLP-induced, but not histamine-induced, oedema formation. This effect was independent of cyclo-oxygenase inhibition and the drug did not induced neutropenia. 7. Intravenous injection of the microtubule blocking agent colchicine inhibited FMLP-induced oedema formation. Responses to bradykinin were unaffected. When colchicine was administered after intradermal FMLP, subsequent plasma leakage was abolished. 8. The inference that receptors have evolved to bacterial secretions (i.e. FMLP) and products of the interaction of bacterial cell walls with tissue fluid (i.e. C5a des Arg), is consistent with the hypothesis that oedema formation is fundamentally a functional process concerned with regulating microbial lysis and opsonisation in an infected tissue.

Animals↗
PubMed1993

Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505.

1. The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505. 6. In vitro, UK-74,505 inhibited aggregation and the increase in intracellular calcium concentration induced by PAF in rabbit washed platelets in a concentration-dependent manner (IC50 = 1.6 x 10-8 M and 1.1 x 10-8 M, respectively). Platelets pretreated with 10-6 M UK-74,505, and maintained at 37 degrees C,were unresponsive to PAF, whilst responding normally to thrombin, for up to 4 h.7. In a second series of in vivo experiments, platelets were labelled with 111In and loaded with Fura-2.The platelets were then pretreated with 10-6 M UK-74,505, washed, and injected into recipient rabbits.These platelets, prepared from blood samples taken at the end of the in vivo experiments, exhibited an 80% reduction in their response to PAF as measured ex vivo with Fura-2. However, in contrast to the effects of i.v. UK-74,505, platelets pretreated with the antagonist did accumulate effectively in the RPA reaction, a significant reduction only being observed in responses at the lowest antibody dose. In addition, pretreatment of platelets had no effect on the small platelet accumulation induced by PAF.8. These results suggest that PAF is an important mediator of oedema formation and platelet accumulation in the RPA reaction in rabbit skin. However, they question the role of PAF receptors on platelets in this model. The results also indicate that PAF may be involved in platelet accumulation induced by zymosan in rabbit skin.

Animals↗
PubMed1993

Suppression by intradermal administration of heparin of eosinophil accumulation but not oedema formation in inflammatory reactions in guinea-pig skin.

1. Heparin is widely used in the treatment of thrombotic disorders and as an aid in surgery. Anti-inflammatory effects of heparin have also been described. In this study, we have investigated the effects of locally-injected heparin on the oedema formation and eosinophil accumulation induced by various inflammatory stimuli in guinea-pig skin. 2. Heparin dose-dependently suppressed the accumulation of 111In-labelled eosinophils induced by i.d. injection of zymosan-activated plasma (ZAP). The 111In-eosinophil accumulation induced by other inflammatory stimuli (compound 48/80, platelet activating factor, interleukin-8 and in a passive cutaneous anaphylaxis reaction) was also suppressed by locally-injected heparin. 3. Oedema formation in response to these same stimuli was not altered by the local injection of heparin. 4. Fucoidin, a negatively-charged sulphated algal polymer, had no effect on the 111In-eosinophil accumulation or oedema formation induced by ZAP. Nevertheless, fucoidin significantly suppressed the oedema formation induced by i.d. injection of cationic protein-containing extracts of Schistosoma mansoni larvae. Heparin also inhibited oedema induced by the extracts, suggesting that both fucoidin and heparin were effectively neutralizing the cationic protein of the extracts to inhibit their oedema-inducing activity. 5. Thus, heparin significantly inhibited the accumulation of 111In-eosinophils, but not oedema formation, induced by various inflammatory stimuli. This, taken together with the lack of effect of fucoidin, suggests that heparin interferes with the process of eosinophil trafficking by a mechanism that does not depend on neutralisation of the charge of the stimulatory molecules.

Administration, Cutaneous↗
PubMed1994

Differential effects of the PAF receptor antagonist UK-74,505 on neutrophil and eosinophil accumulation in guinea-pig skin.

1. The effect of the dihydropyridine, platelet activating factor (PAF) receptor antagonist, UK-74,505, on leucocyte accumulation and oedema formation in guinea-pig skin was investigated. The inflammatory reactions studied were elicited by exogenous mediators, a passive cutaneous anaphylactic (PCA) reaction and zymosan particles. 2. Leucocyte accumulation and oedema formation were measured as the local accumulation of i.v. administered 111In-labelled neutrophils or eosinophils together with 125I-labelled albumin. UK-74,505 was either administered i.v. or used to pretreat the radiolabelled leucocytes in vitro prior to their last wash and injection into recipient animals. 3. In vitro, UK-74,505 inhibited PAF-induced elevations in cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2-loaded guinea-pig neutrophils and eosinophils with IC50 values of 10(-9) M and 7 x 10(-9) M respectively. Neutrophils and eosinophils pretreated with 10(-7) M and 10(-6) M UK-74,505 respectively, and maintained at 37 degrees C, were unresponsive to PAF for the 4 h period investigated. 4. In vivo, using 2 h test periods, i.v. UK-74,505 (0.5 and 2.5 mg kg-1) inhibited the accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation induced by intradermal PAF, but had no effect on responses elicited by leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP, used as a source of C5a des Arg). UK-74,505 (2.5 mg kg-1) was also without an effect on response induced by a PCA reaction but significantly suppressed the 111In-eosinophil accumulation following the intradermal administration of zymosan particles. The 111In-neutrophil accumulation induced by zymosan particles was not, however, affected by UK-74,505. 5. In a second series of in vivo experiments, "'In-leucocytes were pretreated in vitro with UK-74,505 prior to their last wash and injection into recipient animals. Radiolabelled neutrophils, and eosinophils were pretreated with 10-7 M and 10-6 M UK-74,505 respectively, concentrations previously shown to block the leucocyte responses to PAF in vitro for up to 4 h. The in vitro pretreatment of the cells with the PAF antagonist, whilst not affecting the responses to intradermally-injected PAF, suppressed the"'In-eosinophil accumulation response induced by zymosan particles.6. The results of this study indicate that PAF is not involved in neutrophil accumulation, eosinophil accumulation and oedema formation induced by LTB4, ZAP and a PCA reaction. Endogenous PAF does, however, appear to have a role in zymosan-induced eosinophil accumulation but not neutrophil accumulation, suggesting the existence of different inflammatory pathways in the induction of neutrophil and eosinophil accumulation in vivo. Furthermore, while leucocyte accumulation induced by exogenous PAF does not appear to involve leucocyte PAF receptors, the mechanism by which endogenous PAF mediates the zymosan-induced eosinophil accumulation appears dependent on the expression of PAF receptors on eosinophils.

Animals↗
PubMed1994

Studies on the mechanisms involved in the inflammatory response in a reversed passive Arthus reaction in guinea-pig skin: contribution of neutrophils and endogenous mediators.

1. Mediators of inflammation can increase vascular permeability in at least two different ways: by acting directly on endothelial cells or, indirectly, through an incompletely understood mechanism, dependent on circulating neutrophils. Neutrophil-dependent oedema formation has been described in the skin of rabbits, rats, hamsters, mice and man. In contrast, we presented evidence in a previous study that local oedema formation induced by i.d. injection of chemoattractants in guinea-pig skin was neutrophil-independent. In the present study, we sought evidence of neutrophil-dependent oedema formation in immune-complex-mediated vasculitis, the reversed passive Arthus (RPA) reaction, in guinea-pig skin. We also investigated whether haemorrhage in the RPA reaction was neutrophil-dependent (as it is in other species) and the role of endogenous mediators of inflammation (prostaglandins, nitric oxide, histamine, PAF and leukotrienes) in contributing to the local inflammatory response. 2. In the RPA reaction, most oedema formation occurred over the first 60 min whereas 111In-neutrophil accumulation was still increasing from 60 to 240 min. The different kinetics of these two events suggested that they may be dissociated. 3. Oedema formation was partially inhibited by a long-acting PAF antagonist (UK-74,505) and an H1 histamine receptor antagonist (mepyramine) but not by a 5-lipoxygenase inhibitor (ZM 230487). A nitric oxide synthesis inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) suppressed oedema formation by 68% whereas a cyclo-oxygenase inhibitor suppressed oedema by 27%. 4. 111In-neutrophil accumulation in the RPA reaction was partially suppressed by UK-74,505. In contrast, ZM 230487 was without effect at doses which abrogated arachidonic acid-induced 111In-neutrophil accumulation. 5. The anti-CD18 monoclonal antibody, (mAb) 6.5E F(ab')2, effectively inhibited 111In-neutrophil accumulation induced by PAF, zymosan-activated plasma (ZAP) and in the RPA reaction. However, oedema formation measured in the same sites was not altered. In contrast, oedema formation in the RPA reaction was partially suppressed by 6.5E whole mAb which was 2.5 times more potent than 6.5EF(ab')2 at inhibiting guinea-pig neutrophil adhesion to protein-coated plastic. Haemorrhage induced by PAF and in the RPA reaction was significantly inhibited by 6.5E F(ab')2 pretreatment.6. We conclude that in the RPA reaction in guinea-pig skin, oedema formation is partially neutrophil dependent as assessed by using an anti-CD18 mAb, whereas ZAP-induced oedema formation is neutrophil-independent. Haemorrhage was also dependent on neutrophil accumulation. In addition, our studies support a role for PAF in mediating both oedema formation and "'In-neutrophil accumulation in the RPA reaction. Endogenous release of histamine also appears to be important in mediating oedema formation suggesting that mast cells play a critical role in increases of vascular permeability in inflammatory reactions in guinea-pig skin. Moreover, our results confirm previous findings which suggest a dominant role for nitric oxide in maintaining cutaneous blood flow in the guinea-pig.

Animals↗
PubMed1990

Cloning and characterization of T-cell-reactive protein antigens from Listeria monocytogenes.

To explore the molecular basis of the T-cell-mediated immune response to Listeria monocytogenes, we cloned and expressed listerial antigens in Escherichia coli using the lambda-ZAP bacteriophage and Bluescript plasmid vectors. A two-stage screening strategy was implemented to identify T-cell-reactive antigens; the first stage involved antibodies or oligonucleotide probes and the second stage was based on assays for T-cell activation. A library of genomic DNA from L. monocytogenes was generated in lambda-ZAP, and then antigens, were detected in infected cells with a polyclonal rabbit anti-L. monocytogenes antiserum and an L. monocytogenes-specific monoclonal antibody. Also, synthetic oligonucleotide probes corresponding to the structural gene for listeriolysin O (LLO) were used to screen the recombinant DNA library. In each case, positive isolates were evaluated for T-cell antigenicity by measuring antigen-induced interleukin-2 production by polyclonal T cells taken from L. monocytogenes-immune mice. Phage clones were subcloned and expressed in the Bluescript plasmid and tested further for antigenic activity and LLO expression. Using this screening strategy, we successfully identified bacterial clones producing recombinant listerial antigens which activate L. monocytogenes-immune T cells in vitro. Antigens operative in the T-cell response during infection with L. monocytogenes include LLO, 62- and 39-kilodalton proteins, and other poorly defined bacterial surface components. We also found that high concentrations of recombinant LLO inhibited macrophage-mediated antigen presentation. These results are discussed in terms of the multiple functions of LLO as a virulence factor, inhibitor of antigen presentation, and potent antigen in the T-cell response to L. monocytogenes. These studies represent the first step toward a genetic definition of the antigens recognized in immune defense to L. monocytogenes.

Animals↗
PubMed2006

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype.

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage lambda ZAP Express vector which contains both prokaryotic (P(lac)) and eukaryotic (P(CMV)) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which P(CMV)-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into lambda ZAP Express, and two strongly immunodominant clones, lambda-A8 and lambda-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone lambda-A8 expressed an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone lambda-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones lambda-A8 and lambda-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone lambda-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone lambda-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Animals↗
PubMed1988

Selective lung leukosequestration after complement activation.

This study tests whether activated complement leads to a selective entrapment of polymorphonuclear leukocytes (PMN's) in the lungs. Awake sheep were infused for 5 min with zymosan-activated plasma (ZAP, 2.5 mg/ml) at a rate of 5 ml/min into the superior vena cava (IV, n = 4) or intra-arterially into the aortic arch or femoral artery (IA, n = 8). At the end of IV infusion, leukocyte counts fell from 8,862 to 1,631/mm3 (P less than 0.01). PMN counts across the lungs decreased by 74%. There were increases in plasma thromboxane (Tx) B2 from 114 to 2,733 pg/ml (P less than 0.01), mean pulmonary arterial pressure from 12 to 42 mmHg (P less than 0.01), and physiological shunt from 13 to 25% (P less than 0.05). Within 1 h lymph TxB2 levels had risen from 301 to 4,916 pg/ml (P less than 0.01), lung lymph flow (QL) rose from 3.7 to 11.1 ml/30 min (P less than 0.05), lymph-to-plasma protein ratio (L/P) remained unchanged at 0.63, and lymph protein clearance increased from 2.3 to 7.5 ml/30 min (P less than 0.05). Leukosequestration, quantitated by capillary PMN counting and by assaying the granulocyte marker myeloperoxidase, occurred relative to sham animals (P less than 0.05) in the lung and spleen but not in other organs. Intra-arterial ZAP infusion led to changes that were similar in magnitude and timing to the IV group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Zymosan-activated plasma causes prolonged decreases in PMN superoxide release in sheep.

It is well established that activation of neutrophils within the pulmonary circulation produces acute lung injury in which adherence of neutrophils to endothelial cells is an obligatory step in the mechanism of injury. The effects of in vivo activation of neutrophils on the in vitro responses of these cells to stimulation have not been determined, although such information may be important in understanding how different etiological factors may interact to produce infection or acute respiratory failure. By using an assay to sequentially measure superoxide anion (O2-) release from adherent neutrophils stimulated with phorbol myristate acetate (PMA), we measured the in vitro activation response of peripheral blood neutrophils isolated before and 24 h after infusion of zymosan-activated plasma (ZAP; or untreated plasma as a control), air bubbles, or PMA in awake, instrumented sheep. Each of the three inflammatory agents produced an increase in lung microvascular permeability characteristic of acute lung injury; control plasma did not. For the in vivo ZAP experiments, stimulated O2- release in vitro by using PMA was approximately 50% lower (P less than 0.05) for neutrophils isolated 24 h after the in vivo infusion (4.3 +/- 0.8 nmol/500,000 cells) than before (8.1 +/- 0.2 nmol/500,000 cells). For the air emboli or PMA in vivo experiments, there were no changes in neutrophil activation responses in vitro. Similarly, infusion of control plasma did not result in reduced neutrophil O2- release. These results show that alterations in the inflammatory potential of neutrophils may occur in vivo and that such alterations appear to be dependent on the mechanism and agent by which lung injury is produced.

Animals↗
PubMed1996

Subthreshold membrane resonance in neocortical neurons.

1. Using whole cell recording techniques, we studied subthreshold and suprathreshold voltage responses to oscillatory current inputs in neurons from the sensorimotor cortex of juvenile rats. 2. Based on firing patterns, neurons were classified as regular spiking (RS), intrinsic bursting (IB), and fast spiking (FS). The subthreshold voltage-current relationships of RS and IB neurons were rectifying whereas FS neurons were almost ohmic near rest. 3. Frequency response curves (FRCs) for neurons were determined by analyzing the frequency content of inputs and outputs. The FRCs of most neurons were voltage dependent at frequencies below, but not above, 20 Hz. Approximately 60% of RS and IB neurons had a membrane resonance at their resting potential. Resonant frequencies were between 0.7 and 2.5 Hz (24-26 degrees C) near -70 mV and usually increased with hyperpolarization and decreased with depolarization. The remaining RS and IB neurons and all FS neurons were nonresonant. 4. Resonant neurons near rest had a selective coupling between oscillatory inputs and firing. These neurons selectively fired action potentials when the frequency of the swept-sine-wave (ZAP) current input was near the resonant frequency. However, when these neurons were depolarized to -60 mV, spike firing was associated with many input frequencies rather than selectively near the resonant frequency. 5. We examined three subthreshold currents that could cause low-frequency resonance: IH, a slow, hyperpolarization-activated cation current that was blocked by external Cs+ but not Ba2+; IIR, an instantaneously activating, inwardly rectifying K+ current that was blocked by both Cs+ and Ba2+; and INaP, an quickly activating, inwardly rectifying persistent Na+ current that was blocked by tetrodotoxin (TTX). Voltage-clamp experiments defined the relative steady state activation ranges of these currents. IIR (activates below -80 mV) and INaP (activates above -65 mV) are unlikely to interact with each other because their activation ranges never overlap. However, both currents may interact with IH, which activated variably at potentials between -50 and -90 mV in different neurons. 6. We found that IH produces subthreshold response. Consistent with this, subthreshold resonance was blocked by external Cs+ but not Ba2+ or TTX. Application of Ba2+ enlarged FRCs and resonance at potentials below -80 mV, indicating that IK,ir normally attenuates resonance. Application of TTX greatly diminished resonance at potentials more depolarized than -65 mV, indicating that INaP normally amplifies resonance at these potentials. 7. The ZAP current input may be viewed as a model of oscillatory currents that arise in neocortical neurons during synchronized activity in the brain. We propose that the frequency selectivity endowed on neurons by IH may contribute to their participation in synchronized firing. The voltage dependence of the frequency-selective coupling between oscillatory inputs and spikes may indicate a novel mechanism for controlling the extent of low-frequency synchronized activity in the neocortex.

Animals↗
PubMed1986

Thromboxane as a mediator of pulmonary dysfunction during intravascular complement activation in sheep.

Intravascular complement activation results in thromboxane (TxA2) production, pulmonary hypertension, hypoxemia, and increased lung vascular permeability. The purpose of this study was to determine the role of TxA2 as a mediator of these responses. Experiments were made in anesthetized sheep subjected to intravenous injections of zymosan-activated plasma (ZAP) every 30 min for 4 h. Sheep were pretreated with dazoxiben, a TxA2 synthetase inhibitor, or SK and F 88046, a TxA2 end-organ antagonist, and the results were compared with those from untreated sheep. Dazoxiben, but not SK and F 88046, inhibited TxA2 release. The hypertensive response averaged 74 +/- 3 cm H2O after each injection of ZAP in untreated sheep. Neither drug altered this response. Pao2 decreased an average of 20 +/- 1 mmHg in untreated sheep, 3 +/- 1 mmHg in dazoxiben-treated sheep, and 11 +/- 1 mmHg in SK and F 88046-treated sheep. Increases in lung lymph flow and lymph protein clearance were unaffected by treatment. TxA2 appears to be an important mediator of hypoxemia during intravascular complement activation.

6-Ketoprostaglandin F1 alpha↗
PubMed1979

Corticosteroids inhibit complement-induced granulocyte aggregation. A possible mechanism for their efficacy in shock states.

Granulocyte (PMN) aggregation and embolization may underlie complement (C)-mediated organ dysfunction in such syndromes as hemodialysis neutropenia and Purtscher's ischem;c retinopathy. Because of clinical and pathologic parallels, we have further suggested a role for this phenomenon in the genesis of the adult respiratory distress syndrome (ARDS). Because corticosteroids are commonly used in immune diseases, and have particularly been claimed efficacious in shock and ARDS, we tested the capability of methylprednisolone (MP), hydrocortisone (HC), and dexamethasone (DEX) to inhibit PMN aggregation. Aggregation engendered in vitro by zymosan-activated plasma (ZAP) was inhibited by MP and HC at concentrations approximating plasma levels achieved with the large bolus (30 mg/kg i.v) therapy advocated in shock states; DEX was almost without effect. Using intravital fluorescence microscopy, we observed PMN aggregation and embolization in the mesenteric vessels of rats given intra-arterial infusions of ZAP; this was also prevented by pretreatment with 30 mg/kg MP. Steroid inhibition of aggregation seemed not to involve disruption of receptor function, because aggregation induced by alternative agents, n-formyl-Met-Leu-Phe and the ionophore A23187, was also inhibited by MP. Moreover, corticosteroid inhibition of PMN prostaglandin synthesis is also an unlikely explanation for our results, since aspirin and ibuprofen failed to block aggregation and arachidonic acid neither effected aggregation itself nor ameliorated the steroid effect. Our studies provide a plausible rationale for the empiric observation that high-dose corticosteroids may benefit patients with syndromes associated with microvascular leukostasis.

Cell Aggregation↗