The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco. (+info)
Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis.
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We have investigated the wasp venom peptides mastoparan X and polistes mastoparan regarding their apparent potential to induce pore-like defects in phosphatidylcholine unilamellar vesicles. Based on a fundamental theoretical model, the pore activation and deactivation kinetics have been evaluated from the observed efflux of liposome entrapped carboxyfluorescein in relation to the bound peptide to lipid ratio. We can quantitatively describe our experimental data very well in terms of a specific reaction scheme resulting in only a few short-lived pores. They evidently emerge rapidly from a prepore nucleus being produced by two rate-limiting monomeric states of bound peptide. These peculiar states would be favorably populated in an early stage of bilayer perturbation, but tend to die out in the course of a peptide/lipid restabilization process. (+info)
Glucocorticoids increase the endocytic activity of human dendritic cells.
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We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10(-8) M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC-dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex. The expression of receptors involved in antigen capture was investigated by FACS analysis. Dex up-regulates MR, CD16 and CD32 expression on DC. After maturation with tumor necrosis factor-alpha or CD40 ligand in Dex-treated DC, despite a reduction induced by maturation the endocytic activity of FITC-dextran and LY, the expression of MR, CD16 and CD32 remained higher than in control DC. In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting tetanus toxin to specific autologous T cell lines. In conclusion our data suggest a new mechanism by which GC may influence immune responses. In fact with the increase in endocytic activity, Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibiting the capacity to stimulate T cells. (+info)
Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.
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The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression. (+info)
Negative interstitial pressure in the peritendinous region during exercise.
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In the present study, tissue pressure in the peritendinous area ventral to the human Achilles tendon was determined. The pressure was measured during rest and intermittent isometric calf muscle exercise at three torques (56, 112, and 168 Nm) 20, 40 and 50 mm proximal to the insertion of the tendon in 11 healthy, young individuals. In all experiments a linear significant decrease in pressure was obtained with increasing torque [e.g., at 40 mm: -0.4 +/- 0.3 mmHg (rest) to -135 +/- 12 mmHg (168 Nm)]. No significant differences were obtained among the three areas measured. On the basis of these observations, microdialysis was performed in the peritendinous region with a colloid osmotic active substance (Dextran 70, 0.1 g/ml) added to the perfusate with the aim of counteracting the negative tissue pressure. Dialysate volume was found to be fully restored (100 +/- 4%) during exercise. It is concluded that a marked negative tissue pressure is generated in the peritendinous space around the Achilles tendon during exercise in humans. Negative tissue pressure could lead to fluid shift and could be involved in the increase in blood flow previously noted in the peritendinous tissue during exercise (H. Langberg, J. Bulow, and M. Kjaer. Acta Physiol. Scand. 163: 149-153, 1998; H. Langberg, J. Bulow, and M. Kjaer. Clin. Physiol. 19: 89-93, 1999). (+info)
The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers. The effects of charges and pH.
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The alpha-helix of the designed amphipathic peptide antibiotic LAH(4 )(KKALLALALHHLAHLALHLALALKKA-NH(2)) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768-775). LAH(4) exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of LAH(4) are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH(4) adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH(4) is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH(4) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments. (+info)
Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion.
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We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion. (+info)
The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.
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Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides. (+info)