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D O Haskard

Publications and source records attributed to D O Haskard.

At least 55 records · Page 3Linked to original sources
PubMed2000

An anti-inflammatory property of aprotinin detected at the level of leukocyte extravasation.

BACKGROUND: Aprotinin is a serine protease inhibitor used extensively in cardiac operations to reduce postoperative bleeding. It has also been used in trials aimed at reducing the systemic inflammatory response to cardiopulmonary bypass. It remains unclear whether the anti-inflammatory action of aprotinin is related to its general ability to suppress leukocyte activation or whether aprotinin can exercise effects during the leukocyte-endothelial cell adhesion cascade. METHODS: We used intravital microscopy to study the 3 main stages of the adhesion cascade (leukocyte rolling, firm adhesion, and extravasation) within the mesenteric microcirculation of rats. This in vivo technique allows leukocyte recruitment to be viewed directly through the transparent mesentery of anesthetized animals. RESULTS: Aprotinin, given by continuous infusion at a clinically relevant dose, exerted no effect on the rolling or firm adhesion responses toward local chemoattractant N -formyl-methyl-leucyl-phenylalanine but significantly inhibited extravasation of leukocytes (73% at 40 minutes, P =.04) into surrounding tissues. In parallel in vitro experiments, aprotinin (used at 200, 800, and 1600 kIU/mL) dose dependently inhibited neutrophil transmigration through cultured endothelial cells in response to 3 different chemoattractants: N -formyl-methyl-leucyl-phenylalanine (P <.001 at 800 and 1600 kIU/mL), interleukin 8 (P <.05 at 200 kIU/mL and P <.001 at 800 and 1600 kIU/mL), and platelet-activating factor (P <.05 at 1600 kIU/mL). CONCLUSIONS: Our studies have therefore revealed a novel anti-inflammatory mechanism of aprotinin operating at the level of leukocyte extravasation. These findings may be relevant in the prevention of systemic inflammation after cardiopulmonary bypass through the use of protease inhibitors.

Analysis of Variance↗
PubMed2000

The antithrombotic effect of aprotinin: actions mediated via the proteaseactivated receptor 1.

BACKGROUND: Despite aprotinin being in widespread clinical use to prevent bleeding during cardiac surgery, there remains concern that such a powerful hemostatic agent may also be prothrombotic, particularly in relation to coronary vein graft occlusion. The major thrombin receptor on platelets, protease-activated receptor 1 (PAR1) requires proteolytic cleavage to transmit activating signals. Here we have investigated the effect of aprotinin on thrombin-induced PAR1 activation of platelets. METHODS AND RESULTS: Proteolysis-dependent and -independent responses of washed platelets were studied in vitro. Platelet aggregation induced by trypsin was dependent on PAR1 (inhibited by the PAR1-specific antagonist peptide, FLLRN) and was completely blocked by aprotinin at doses more than 100 KIU/mL. Aggregation in response to thrombin, 1 nmol/L, was predominantly mediated through PAR1 and was inhibited 42.6% to 86.6% (P <.05-.001) by pharmacologic doses of aprotinin (50-160 KIU/mL). Aprotinin did not inhibit the nonproteolytic agonists collagen, epinephrine, adenosine diphosphate, or phorbol 12-myristate 13-acetate. Furthermore, blockade of the thrombin response by aprotinin did not prevent subsequent platelet aggregation through collagen or epinephrine. Experiments with intraplatelet Ca(2+) fluxes, which provided an earlier measure of platelet activation, placed the effect of aprotinin proximal to the PAR1 activation event. Since aprotinin did not inhibit platelet responses to the nonproteolytic PAR1 agonist peptide, SFLLRN, this implied that aprotinin acted by preventing PAR1 receptor cleavage by thrombin. CONCLUSIONS: Aprotinin inhibits thrombin-induced platelet activation by preventing proteolysis of the PAR1 receptor. These findings argue against aprotinin being prothrombotic and suggest instead that aprotinin may have significant antithrombotic effects.

Analysis of Variance↗
PubMed2000

Inhibition of neutrophil L-selectin shedding: a potential anti-inflammatory effect of aprotinin.

The cardiopulmonary bypass (CPB)-related inflammatory response involves leucocyte activation and increased leucocyte-endothelial cell interaction. L-selectin is an adhesion molecule expressed on the surface of leucocytes which participates in the initial rolling step of the leucocyte-endothelial cell adhesion cascade. L-selectin is proteolytically cleaved off the surface of leucocytes when they become activated, an event that is regarded as a marker of leucocyte activation. Aprotinin is a protease inhibitor that has been used in cardiac surgery as a haemostatic agent and also exhibits certain anti-inflammatory properties. In this study, peripheral venous blood from volunteers was pre-incubated with aprotinin at 200, 800 and 1600 kallikrein inhibiting units (kiu)/ml and stimulated with the chemoattractants N-formyl-methyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF). Surface expression of L-selectin on neutrophils was measured using a monoclonal antibody and flow cytometry. The results demonstrate that aprotinin inhibits shedding of L-selectin in a dose-dependent fashion (p=0.0278 and 0.0005, respectively, at 800 and 1600 kiu/ml for fMLP-stimulated shedding; p=0.0017 and 0.0010, respectively, at 200 and 800 kiu/ml for PAF-stimulated shedding). This effect may be of significance with respect to the anti-inflammatory action of aprotinin in patients undergoing CPB.

Aprotinin↗
PubMed2000

Resting and activated T cells induce expression of E-selectin and VCAM-1 by vascular endothelial cells through a contact-dependent but CD40 ligand-independent mechanism.

This study explored the effect on endothelial cell (EC) activation of contact with T lymphocytes, which occurs during lymphocyte emigration into inflamed tissues. Addition of T cells to umbilical vein or dermal microvascular EC monolayers stimulated expression of EC E-selectin and VCAM-1. This response required direct cell:cell contact, but not T-cell activation. The capacity of resting CD4+ T cells to activate EC was restricted to the CD45RO+ subset and could be enhanced by 6 h prestimulation of T cells with PMA and ionomycin. The EC-stimulating capacity of resting or activated T cells was independent of CD40 ligand. Furthermore, inhibition of TNF-alpha/beta and IL-1alpha/beta, together with CD40 ligand, failed to inhibit EC activation by resting T cells and only inhibited the response to PMA- and ionomycin-activated T cells by 40 +/- 18%. Our data suggest that T-cell-EC interactions can lead to EC activation through a novel contact-dependent, but CD40 ligand-independent, mechanism.

CD40 Antigens↗
PubMed1999

Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence.

The selectins are adhesion molecules that mediate the tethering and rolling of leukocytes on vascular endothelium. Although E-selectin and P-selectin are known to be expressed by endothelial cells (EC) in response to proinflammatory stimuli, their pattern and mechanisms of expression in immune-mediated inflammation remain poorly understood. By quantifying luminal endothelial selectin expression via i.v. administration of radiolabeled mAb, we detected constitutive expression of P-selectin, but not E-selectin, in mouse skin. Both selectins were transiently up-regulated after intradermal TNF-alpha, IL-1alpha, or IL-1beta. In contrast, during a contact sensitivity response to oxazolone, expression of both selectins was prolonged, with distinct peaks at 6 and 48 h. Experiments with P-selectin gene-targeted mice showed that the P-selectin measured was exclusively expressed by EC rather than platelets. The early and late phases of selectin expression in contact sensitivity were differentiated in terms of their requirement for prior sensitization, and the action of IL-1. Whereas the early phase was a nonspecific 'irritant' response to oxazolone, the late phase was Ag specific and was partially IL-1 dependent. Therefore, persistence of both E- and P-selectin expression in vivo can occur as a result of sequential and distinct EC activation processes that appear to be at least partially different from those previously reported as stimulating ICAM-1 and VCAM-1 expression. The further elucidation of mechanisms of EC activation in this model may help determine the relative roles of selectins and ligands for leukocyte integrins in the sequential recruitment of T cells and other leukocyte subsets during ongoing immune-mediated inflammatory responses.

Animals↗
PubMed1999

TNF-alpha and IL-1 sequentially induce endothelial ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice.

Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-alpha and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-alpha by 14 wk, with levels of circulating IL-1alpha and IL-1beta being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-alpha antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-alpha and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-alpha is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.

Animals↗
PubMed1999

Induction of decay-accelerating factor by cytokines or the membrane-attack complex protects vascular endothelial cells against complement deposition.

Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

Antigens, CD↗
PubMed1999

Repetitive myocardial stunning in pigs is associated with the increased expression of inducible and constitutive nitric oxide synthases.

OBJECTIVES: Nitric oxide (NO) has complex effects on myocardial function particularly following ischaemia-reperfusion. The goal of this study was to examine the result of repetitive myocardial stunning on myocardial NO release and expression of inducible (iNOS) and constitutive (eNOS) NO synthases. METHODS AND RESULTS: Propofol anaesthetised pigs underwent ten, 2-min episodes of circumflex artery occlusion (n = 6) or acted as sham operated controls (n = 4). Measurements of segment shortening demonstrated a fall in function in the ischaemic territory to 52.5 +/- 7.3% (mean +/- S.E.M.) of baseline shortening 30 min after the stunning stimulus, recovering to 92 +/- 8.7% 5.5 h later. Function remained stable in sham controls. The change in venous-arterial [NO] between baseline and 6 h reperfusion was found to be significantly different between the two groups (0.2 +/- 0.7 in stunned vs. -4.3 +/- 1.6 microM in shams; P < 0.02). Western blotting and band optical density used to compare tissue from stunned territory (S), non-stunned territory (IC) and sham control animals (SC) demonstrated this was associated with an increase in the expression of both iNOS (S: 93 +/- 13.4, IC: 37 +/- 2.4 and SC: 25 +/- 4 [arbitrary units], P < 0.01 and P = 0.031) and eNOS (S: 104 +/- 7.4, IC; 62.5 +/- 7.4 and SC; 75.7 +/- 0.6, P < 0.03 and P < 0.01) in stunned myocardium. Immunocytochemistry localised iNOS reactivity to vascular smooth muscle cells and cardiomyocytes in stunned tissue and eNOS reactivity to endothelial cells. CONCLUSION: Recovery from repetitive myocardial stunning is associated with the increased expression of both iNOS and eNOS and would be compatible with a protective role for both these enzymes. This finding has possible relevance for both the late window of ischaemic preconditioning and myocardial hibernation.

Animals↗
PubMed1999

Endothelial expression of VCAM-1 in experimental crescentic nephritis and effect of antibodies to very late antigen-4 or VCAM-1 on glomerular injury.

The migration of leukocytes into glomeruli in crescentic glomerulonephritis is fundamental to pathogenesis, and offers important therapeutic opportunities. We addressed the importance of VCAM-1, and its leukocyte ligand very late antigen-4 (VLA-4), in such leukocyte migration. In a rat model of nephrotoxic nephritis, glomerular expression of VCAM-1, studied by immunohistochemistry, was up-regulated by day 6 of nephritis. To quantify kidney endothelial VCAM-1 expression, a differential radiolabeled mAb technique was used, which demonstrated that protein expression was not up-regulated by day 2 of nephritis, but rose threefold between days 2 and 5, and remained elevated until at least day 28. An in vivo study was then performed, using blocking mAbs to either VCAM-1 or VLA-4, starting mAb treatment on the day prior to disease induction, and continuing until animals were sacrificed at day 7. mAbs to VLA-4 significantly attenuated renal injury (albuminuria, glomerular fibrinoid necrosis, and crescent formation), but mAbs to VCAM-1 had no significant effect. Surprisingly, the number of leukocytes within glomeruli was unaffected by anti-VLA-4 mAb therapy, despite the reduction in renal injury. Paradoxically, classical markers of macrophage activation were increased in the anti-VLA-4- and anti-VCAM-1-treated animals. This study demonstrates that kidney endothelial VCAM-1, in contrast to ICAM-1, is not up-regulated by day 2 of nephrotoxic nephritis, and plays little part in early leukocyte influx into glomeruli. However, VLA-4 is an important mediator of glomerular injury, operating after transendothelial leukocyte migration, and presumably binding to alternate ligands within the kidney.

Albuminuria↗
PubMed1999

A method for determining the cytoprotective effect of catalase in transiently transfected cell lines and in corneal tissue.

Both when developing gene constructs for therapeutic purposes and when testing the biological function of proteins, it would be convenient to use cells or tissues that have been transiently transfected with the gene of interest. However, determining the protective effects of transient gene expression is complicated by a low transfection efficiency, resulting in only a minority of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this study we have developed a convenient technique for determining cell death in transiently transfected vascular endothelial cell monolayers and in corneal tissue. Vascular endothelial cells were cotransfected with human catalase cDNA and the lacZ gene encoding beta-galactosidase, under conditions in which cells expressing beta-galactosidase also expressed catalase. By assaying release of beta-galactosidase upon cell death, it was possible to show that catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was adapted to demonstrate the protective effects of catalase transfection on hydrogen peroxide-mediated injury of intact corneal endothelium under ex vivo culture conditions. This assay should also be useful for characterizing the cytoprotective effects of other genes in transient transfection systems.

Animals↗
PubMed1999

Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity in mice: evidence for a dominant role of TNF-alpha.

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.

Animals↗
PubMed1999

MRI in neuro-Behçet's syndrome: comparison of conventional spin-echo and FLAIR pulse sequences.

We compared the sensitivity of a fluid-attenuated inversion-recovery (FLAIR) sequence with that of a conventional dual-echo spin-echo (SE) sequence) to brain lesions in 20 patients with Behçet's syndrome. They underwent 25 MRI examinations. The images were independently analysed for the number, type and anatomical location of lesions shown. There were 18 abnormal studies (13 initial and 5 follow-up). The FLAIR sequence detected significantly more lesions than the SE TE 80 (P < 0.05) and SE TE 20 (P < 0.01) sequences. It was particularly useful for demonstrating lesions in the juxtacortical white matter, which accounted for over half the lesions detected on the FLAIR images. Of patients presenting with nonspecific symptoms such as headache, seven had normal and five had abnormal studies. All patients presenting with focal neurological signs had abnormal imaging. We found supratentorial and, in particular, juxtacortical lesions to be more frequent than previously described.

Adult↗
PubMed1999

Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin.

We have aimed at selective gene delivery to vascular endothelial cells (EC) at sites of inflammation, by targeting E-selectin, a surface adhesion molecule that is only expressed by activated EC. An anti-E-selectin mAb, 1.2B6, was complexed with the adenovirus vector AdZ.FLAG (expressing the FLAG peptide) by conjugating it to an anti-FLAG mAb. Gene transduction of cultured EC was increased 20-fold compared with AdZ.FLAG complexed with a control bsAb providing EC were activated by cytokines. The anti-E-selectin-complexed vector transduced 29 +/- 9% of intimal EC in segments of pig aorta cultured with cytokines ex vivo, compared with less than 0.1% transduced with the control construct (P < 0.05). This strategy could be developed to target endothelium in inflammation with genes capable of modifying the inflammatory response.

Adenoviridae↗
PubMed1999

VCAM-1 contributes to rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4: evidence for basal expression of functional VCAM-1 in rat skin.

The aim of the present study was to investigate the role of the adhesion pathway alpha4 integrins/vascular cell adhesion molecule type 1 (VCAM-1) in rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4. For this purpose we have used an in vivo model of local 111In-eosinophil accumulation to quantify eosinophil accumulation induced by intradermal administration of platelet-activating factor (PAF) and leukotriene B4 (LTB4) in rats. Initial experiments carried out over 4 hr demonstrated that intravenous administration of an anti-VCAM-1 monoclonal antibody (mAb; 5F10) or an anti-alpha4 integrin mAb (TA2) caused a significant reduction in PAF- or LTB4-induced 111In-labelled eosinophil accumulation. Time-course experiments demonstrated that the anti-VCAM-1 mAb was effective at suppressing early phases of the 111In-labelled eosinophil accumulation induced by PAF and LTB4 (e.g. within the first 60 min). In contrast, 111In-labelled eosinophil accumulation induced by these chemoattractants was unaffected by the local administration of the transcriptional inhibitor actinomycin D, suggesting a role for basally expressed VCAM-1. Indeed, basal expression of VCAM-1 in rat skin sites was demonstrated by the localization of intravenously administered radiolabelled mAb. The localization of the radiolabelled antibody was not altered in skin sites injected with PAF or LTB4. Finally, the inhibitory effects seen with the anti-VCAM-1 mAb were enhanced when the antibody was co-injected into rats with an anti-intercellular adhesion molecule-1 (ICAM-1) mAb (1A29). The combination of these two mAb also caused a significant inhibition of PAF-induced oedema, as quantified by the local accumulation of 125I-labelled human serum albumin. The results indicate a role for alpha4 integrins/VCAM-1 and ICAM-1, in PAF- and LTB4-induced eosinophil accumulation in vivo and suggest that basally expressed VCAM-1 may have a functional role in rapid accumulation of eosinophils induced by chemoattractants.

Animals↗
PubMed1999

High-density lipoproteins differentially modulate cytokine-induced expression of E-selectin and cyclooxygenase-2.

Atherogenesis is a multifactorial chronic inflammatory disease in which low plasma levels of HDLs are a strong predictor of the condition. Although the mechanism of protection by HDLs is not precisely known, HDLs have been shown to influence many of the events involved in the development of atherosclerosis. Previously we have shown that HDLs inhibited the cytokine-induced expression of adhesion molecules (E-selectin, VCAM-1, and ICAM-1) by endothelial cells (ECs). As the complete transcriptional regulation of all 3 genes requires the NF-kappaB family of transcription factors, we examined the effect of HDLs on activation of NF-kappaB. We also investigated the effect of HDLs on 2 other cytokine-induced genes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclooxygenase (Cox-2; prostaglandin H2 synthase, EC 0.1.14.99.1). E-selectin expression in response to tumor necrosis factor-alpha (TNFalpha) was, as expected, inhibited in ECs that had been preincubated with HDLs. However, the level of secretion of GM-CSF in the same cultures was no different from control. In a similar manner, although HDLs had no effect on steady-state mRNA levels of GM-CSF, the levels of E-selectin were significantly inhibited by HDLs. In transient cotransfection experiments we found that HDLs inhibited the cytokine-induced expression of a reporter gene driven by the E-selectin proximal promoter (-383 to 80) but had no effect on the expression of a reporter gene driven under the control of the proximal promoter of GM-CSF (-627 to 28). As would be predicted from this differential response, HDLs did not influence the nuclear translocation or DNA binding of NF-kappaB, or alter the kinetics of degradation and resynthesis of the inhibitory protein IkappaBalpha. We found that HDLs synergized with cytokine to enhance the expression of Cox-2 and induce the synthesis of its main EC product, prostacyclin (PGI2), a potent inhibitor of platelet and leukocyte functions. In conclusion, HDL induces an antiinflammatory phenotype in cytokine-induced ECs, synergizing with cytokine to induce elevation of Cox-2 in addition to inhibiting adhesion molecule expression. Our studies show that these differential effects are mediated in a manner that is likely to be independent of NF-kappaB per se.

Adult↗
PubMed1999

Pulmonary granulocyte kinetics in relation to endothelial and granulocyte activation.

The aim of the study was to measure the peripheral blood levels of soluble E-selectin in patients with systemic inflammation and compare them with in vivo granulocyte activation, pulmonary intravascular granulocyte pooling, pulmonary extravascular granulocyte migration and 99mTc-diethylenetriaminepenta-acetic acid (DTPA) aerosol clearance, an index of lung injury. The level of soluble E-selectin was measured by capture ELISA. Granulocytes were labelled with 111In and 99mTc for quantification of pulmonary granulocyte kinetics. The pulmonary vascular granulocyte pool (PGP) was expressed as a fraction of the total blood granulocyte pool. Pulmonary granulocyte migration was quantified on 24-h images using the 111In signal. Granulocyte activation was quantified as the percentage of circulating cells showing shape change ('primed'). Lung injury was assessed from the clearance rate of inhaled 99mTc-DTPA aerosol. Eighteen patients with systemic inflammation were studied: five with inflammatory bowel disease, eight with systemic vasculitis, four with graft versus host disease and one with a recent renal transplant. The peripheral blood levels of soluble E-selectin were significantly elevated in patients with systemic inflammation. The level of soluble E-selectin showed a significant association with granulocyte migration (Spearman rank correlation coefficient, Rs=0.53; P<0.05) but not with PGP or with the percentage of cells showing shape change (P>0.05 for both). Granulocyte migration was bimodal: patients were therefore subdivided into 'migrators' and 'non-migrators'. Soluble E-selectin level, 99mTc-DTPA clearance and PGP, but not the percentage of cells showing shape change, were significantly higher in migrators than in non-migrators. We conclude that pulmonary intravascular granulocyte pooling is increased in the presence of increased numbers of circulating primed granulocytes but increased pooling does not by itself promote granulocyte migration into the lung interstitium. Insofar as an elevated level of E-selectin in peripheral blood reflects vascular endothelial activation, the data are consistent with the notion that pulmonary endothelial activation is required, in addition to granulocyte activation and an expanded PGP, for granulocyte migration into lung parenchyma and, therefore, for lung injury to occur.

Adult↗
PubMed1998

Expression of recombinant anti-E-selectin single-chain Fv antibody fragments in stably transfected insect cell lines.

We have investigated the possibility of improving the yield of properly folded recombinant single chain Fv fragments (sFv) of an antibody by expressing the protein in stably transfected Drosophila melanogaster SC-2 cells. The DNA encoding the variable regions of the 1.2B6 anti-E-selectin antibody were used to generate a recombinant sFv. This construct was cloned into the pHEN1 vector for expression in Escherichia coli and the pRmHa-3 vector to generate stably transfected Drosophila SC-2 cell lines. Following expression in the bacterial system, and using standard refolding protocols to obtain active material, it was shown that the majority of the sFv formed non-covalent aggregates. In addition SDS-PAGE analysis indicated that even the monomeric material was heterogeneous. In contrast, expression of sFv in Drosophila SC-2 cell lines allowed purification of active sFv directly from the culture supernatant. Only a small proportion of the sFv formed aggregates, and the purified material was homogeneous as determined by SDS-PAGE. Thus the use of stably transfected insect cells has a number of potential advantages in expressing recombinant antibody fragments.

Animals↗