Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells

The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances.     

Mast cell-independent mechanisms of immediate hypersensitivity: a role for platelets

Mast cells have been implicated as the central effectors in allergic responses, yet a fatal anaphylactic response can be induced in mast cell-deficient mice. In this study, we examined the immediate hypersensitivity response in wild-type (WT) and mast cell-deficient mice (W/W(v)) in two different tissues (skin and skeletal muscle). Vascular permeability and leukocyte recruitment were studied after immediate challenge or 4 h postchallenge in OVA-sensitized mice. In skin, immediate challenge induced a significant increase in vascular permeability (75%) within 30 min and was accompanied by increased leukocyte adhesion 4 h postchallenge. In the absence of mast cells, no changes in vascular permeability or leukocyte recruitment were observed in skin. In WT skeletal muscle, immediate challenge induced a rapid increase (80%) in vascular permeability within 5 min and significant leukocyte recruitment after 4 h. Surprisingly, in W/W(v), a gradual increase in vascular permeability was observed, reaching a maximum (50%) within 30 min. Despite the absence of mast cells, subsequent leukocyte emigration was similar to that observed in WT mice. Pretreatment with anti-platelet serum in W/W(v) returned Ag-induced vascular permeability and leukocyte recruitment to baseline. Platelets were shown to interact with endothelium in skeletal muscle, but not dermal microvasculature. These data illustrate that mast cells play a prominent role in vascular permeability and leukocyte recruitment in skin in response to Ag, however, in skeletal muscle; these changes can occur in the absence of mast cells, and are mediated, in part, by the presence of platelets.     

The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice

The role of mast cells in polymorphonuclear leukocyte (PMN) influx in Ag-antibody complex-induced peritonitis was evaluated in mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and their normal littermates, WBB6F1-+/+ (+/+). Peritoneal cell influx was evaluated after i.p. injection of preformed immune complexes. The first significant elevation in the PMN count over PBS-treated controls in +/+ mice was observed 2 h after stimulation. During the period of maximum leukocyte concentrations (6 to 10 h), the increase in total cell count was 5-fold and in PMN 25-fold. In W/Wv mice the PMN influx started 2 h later than in the +/+ mice, and the maximum response (8 to 10 h) was only 50% of that in controls. Reconstitution of mast cells in W/Wv mice for 2 wk or more restored the PMN response to immune complexes. Mast cell release due to AG-antibody complexes was evaluated by measuring fluorescence intensity after berberine sulfate staining for heparin in mast cells from unstimulated as well as stimulated +/+ mice. There was a significant decrease in fluorescence intensity as early as 15 min after stimulation. By 30 min the fluorescence intensity had declined by 65%. This indicates extensive mast cell release that started before PMN mobilization. These experiments demonstrate that mast cells make a significant contribution to immune complex-induced inflammation.     

Neurogenic inflammation, vascular permeability, and mast cells

Electrical stimulation (ES) of sensory nerves causes increased vascular permeability and vasodilatation, a process known as neurogenic inflammation. The purpose of this study was to assess the role of mast cells in neurogenic inflammation induced by ES of sensory nerves. ES of the rat saphenous nerve for 1, 3, 5, 15, or 30 min induced a 166 to 436% increase in the amount of 125I-albumin deposited in the skin. Through the initial 15 min of ES, the histamine content of the skin remained unchanged. However, 30 min of ES caused a 22.1% decrease in skin histamine (p less than 0.05). ES for 5 min followed by measurement of vascular permeability from 0 to 30 min thereafter resulted in maximal increases in 125I-albumin in the skin immediately after cessation of the pulse of ES. When skin histamine was measured at various intervals after a 5-min pulse of ES, no change in the histamine content was observed through the subsequent 30 min. When mast cell degranulation was assessed histologically, 5 min of ES failed to stimulate mast cell degranulation. However, 30 min of ES caused a significant increase in the proportion of degranulating mast cells. When draining venous plasma histamine was monitored before, during and after ES, no change in plasma histamine was observed. In contrast, the intradermal injection of 5 micrograms of compound 48/80 produced a significant increase in plasma histamine. In order to examine the possibility that histamine might be released but remain in the skin after ES, skin "blisters" were developed by intradermal injections of saline. There was a significant increase in the amount of 125I-albumin extravasated into blister fluid measured after 3, 5, and 10 min of ES and a significant increase in histamine after 5 or 10 min. Therefore, prolonged ES of sensory nerves can cause mast cell degranulation. However, ES causes increased vascular permeability at times when no mast cell activation can be observed. These data suggest that the initial phases of neurogenic inflammation are independent of mast cell activation.     

Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17-beta-estradiol induced a concentration- and time-dependent biphasic effect on tight junction. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17-beta-estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17-beta-estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17-beta-estradiol, in line with changes of TER. This study shows that 17-beta-estradiol can induce concentration- and time-related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.     

尼罗罗非鱼消化道肥大细胞的组化性质

实验采用改良甲苯胺蓝(MTB)、阿利新蓝-沙黄(AB/SO)、甲基绿-派洛宁(MG-P)、天青Ⅱ-伊红-瑞氏混合液和硫堇5种组化染色法,对尼罗罗非鱼(Nile tilapia)消化道组织中的肥大细胞(Mast cell,MC)组化性质进行研究。尼罗罗非鱼的食管、胃及小肠壁内均显示有肥大细胞,在食管和胃的切片标本上肥大细胞主要分布在黏膜固有层和胃腺体之间。在肠道中的肥大细胞主要分布在黏膜固有层和肠上皮下方,少量肥大细胞存在于黏膜下层结缔组织中。细胞呈圆形、椭圆形,也有长梭形的。而且肥大细胞有沿血管分布的特点。5种组化染色结果表明:AB/SO、MTB和MG-P显示的MC效果较好,尤其AB/SO染色效果最好,肥大细胞轮廓清楚,胞质颗粒较清晰;尼罗罗非鱼肥大细胞胞浆颗粒都呈红色,即肥大细胞胞浆主要含肝素,不含组胺。天青Ⅱ-伊红-瑞氏混合液染色效果也很好,但被染的肥大细胞较少;80%乙醇硫堇染色,在尼罗罗非鱼消化道各段组织中均未能鉴定出肥大细胞。尼罗罗非鱼消化道肥大细胞大多分布于浅层的黏膜或血管、腺体周围的结缔组织等易表露于环境抗原的位点。罗非鱼消化道黏膜层结缔组织中的肥大细胞与大多数脊椎动物的肥大细胞一样,具有沿血管分布的特性,说明硬骨鱼的肥大细胞如哺乳动物肥大细胞一样与血管有着密切的关系。       

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

Formyl peptide leukocyte chemoattractant uptake and release by cultured human umbilical vein endothelial cells

Recent observations support an active role for the vascular endothelial cell in the induction and evolution of the inflammatory response. Since prior studies suggested that cultured bovine endothelial cells express high affinity binding sites for the neutrophil chemotactic oligopeptide formyl methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), we sought to further characterize the interaction between formyl peptide chemoattractants and human vascular endothelial cells. Cultured human umbilical vein endothelial cells and peripheral blood neutrophils specifically bound f-Met-Leu-[3H]Phe, whereas specific binding to cultured fibroblasts, smooth muscle, and epithelial cells was negligible. Endothelial cells expressed 3.6 +/- 0.7 X 10(5) binding sites/cell with a Kd of 210 +/- 31 nM. Although the hexapeptide formyl norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (f-Nle-Leu-Phe-Nle-Tyr-Lys) and the tetrapeptide f-Met-Leu-Phe-Lys completed with f-Met-Leu-[3H]Phe for binding to endothelial cells, specific binding of 125I-f-Nl-Leu-Phe-Tyr-Lys or f-Met-Leu-Phe-Lys-fluorescein to endothelial cells was not observed, suggesting that steric constraints on formyl peptide binding differ between endothelial cells and leukocytes. At 37 degrees C, cell-associated f-Met-Leu-[3H]Phe greatly exceeded that bound at 0 degrees C and was incorporated predominantly into a nondisplaceable compartment. Release of f-Met-Leu-[3H]Phe or radioactive breakdown products from this compartment was time- and temperature-dependent with a t1/2 of approximately equal to 20 min at 37 degrees C. Resolution of the radioactive products released from f-Met-Leu-[3H]Phe-loaded endothelial cells by thin layer chromatography indicated that greater than or equal to 57% of the released material co-migrated with intact f-Met-Leu-[3H]Phe. Degradative release was blocked by agents that interfere with lysosomal acidification. The radioactive material released from f-Met-Leu-[3H]Phe-loaded endothelial cells bound specifically to neutrophils. This binding was inhibited 50.2 +/- 6.4% by a greater than or equal to 10(3)-fold excess of nonradioactive f-Met-Leu-Phe whereas binding of authentic f-Met-Leu-[3H]Phe was inhibited 89.4 +/- 3.0%. Supernatant obtained from f-Met-Leu-[3H]Phe-loaded endothelial cells elicited a rise in neutrophil cytosolic free calcium ([Ca2+]i) measured by quin2 fluorescence. The change in neutrophil [Ca2+]i depended on ligand binding to the neutrophil formyl peptide receptor since endothelial supernatants were devoid of activity in the presence of the f-Met-Leu-Phe antagonist, tert-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accumulation of platelets at sites of antigen-antibody-mediated injury: a possible role for IgE antibody and mast cells.

Circulating 51Cr-labeled platelets accumulate at skin sites in which a reversed passive Arthus reaction has been induced. The accumulation is biphasic in time and is accompanied by an increased vascular permeability. Increased permeability itself, however, will not produce localization of platelets. A similar platelet accumulation was observed upon injection of compound 48/80 or anti-IgE antibody into the skin and this was not altered in rabbits depleted of complement or neutrophils. Activation of skin mast cells and release of a platelet-activating factor (PAF) is suggested as a mechanism for the effect produced by anti-IgE and compound 48/80. The first phase of platelet accumulation in the Arthus reaction was also unaffected in rabbits depleted of neutrophils or complement, which may suggest a role for IgE antibody and mast cells. The second phase of accumulation was diminished in complement-depleted animals and abrogated in rabbits without neutrophils, suggesting a complement and neutrophil-mediated process but which still might be mediated through mast cell activation by neutrophil cationic protein.     

Vitrification media: toxicity,permeability, and dielectric properties

The aim of this study was to select a cryoprotectant for use in attempts to preserve tissues and organs by vitrification. The first step was to select a cell line with which to compare the toxicity of a range of commonly used cryoprotectants. An immortal vascular endothelial cell (ECV304) was exposed to vitrifying concentrations of four cryoprotectants: dimethyl sulfoxide (Me(2)SO; 45% w/w); 2,3 butanediol (BD; 32%); 1,2-propanediol (PD; 45%); and ethanediol (ED; 45%). Three times of exposure (1, 3, and 9 min) and two temperatures (22 and 2-4 degrees C) were studied. After removal of the cryoprotectant, the ability of the cells to adhere and divide in culture over a 2-day period was measured and expressed as a Cell Survival Index (CSI). There was no measurable loss of cells after exposure to the four cryoprotectants but 3-min exposure to BD, PD, or Me(2)SO at room temperature completely destroyed the ability of the cells to adhere and divide in culture. In contrast, exposure to all four cryoprotectants at 2-4 degrees C for up to 9 min permitted the retention of significant cell function, the CSIs, as a proportion of control, being 76.3+/-7.0% for BD, 63.6+/-7.1% for PD, 37.0+/-4.1 for Me(2)SO, and 33.2+/-3.0 for ED. The permeability properties of the cells for these four cryoprotectants was also measured at each temperature. Permeability to water was high, L(p) approximately equal 10(-7) cm/s/atm at 2-4 degrees C with all the cryoprotectants, but there were substantial differences in solute permeability: BD and PD were the most permeable at 2-4 degrees C (P(s)=4.1 and 3.0 x 10(-6) cm/s, respectively). Equilibration of intracellular cryoprotectant concentration was rapid, due in part to high water permeability; the cells were approximately 80% of their physiological volume after 10 min. Treatment at 2-4 degrees C with BD was the least damaging, but PD was not significantly worse. Exposure to vitrifying concentrations of ED and Me(2)SO, even at 2-4 degrees C, was severely damaging. Segments of rabbit carotid artery were treated with vitrifying concentrations of each of the two most favorable cryoprotectants, BD and PD, for 9 min. It was shown that each cryoprotectant reduced smooth muscle maximum contractility to a similar extent and abolished the acetylcholine response. However, vital staining revealed that exposure to BD also caused substantial damage to the endothelial lining, whereas the endothelium was completely intact after PD exposure, raising the possibility that the effect of PD on NO release may be reversible. In later stages of this project it is planned to use dielectric heating to rewarm the tissues and thereby avoid devitrification. The effects of each cryoprotectant on this mode of heating was therefore studied. Gelatin spheres containing vitrifiable concentrations of each cryoprotectant were rewarmed from -60 degrees C in a radiofrequency applicator. Because the uniformity of heating is related to the dielectric properties of the material, these properties were also measured. PD was the most suitable. These physical measurements, combined with the measurements of toxicity and permeability, indicate that PD is the most favorable cryoprotectant of those tested for use in subsequent stages of this study.     

Mast cells contribute to PACAP-induced dermal oedema in mice.

In the present study the effect of intradermal PACAP-injection on dermal oedema in mice was investigated and the contribution of mast cells to this response was assessed. The injection of PACAP 1-38 into the ears of C57BL/6 mice evoked a dose-dependent response, which, after higher doses of PACAP 1-38, lasted at least 24 h. Histological examination showed significant mast cell degranulation induced by PACAP. Using mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice and the congenic mice, we demonstrated that the the early phase (30 min to 6 h) of PACAP-induced ear swelling response was significantly diminished in mast cell-deficient mice, suggesting that mast cell degranulation contributes to this phase of the response. When mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice were locally and selectively reconstituted by adoptive mast cell transfer, the dermal oedema was almost equal to that of control animals in the early phase of PACAP injection. These results show that mast cell degranulation contributes to PACAP-induced dermal oedema in mice.     

Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.     

Ascorbate protects against vascular leakage in cecal ligation and puncture-induced septic peritonitis

Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128-135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.     

Thermal tolerance during S phase for cell killing and chromosomal aberrations

Synchronous Chinese hamster ovary cells in early S phase were obtained by selecting mitotic cells, accumulating them at the G1/S border by incubating them in aphidicolin for 12 h, and then incubating them for 2 h after releasing them from the aphidicolin block. To determine if thermotolerance could be induced, the cells were heated at 43 degrees C for 20 min in early S phase, incubated for 160 min, and then heated a second time at 43 degrees C for different durations (30-100 min). For the control, nontolerant population, the cells in early S phase were incubated for 50 min and then heated once at 43 degrees C for different durations (20-60 min). Flow cytometric analysis indicated that the population receiving the second heat dose was in the same part of S phase as the population receiving the single heat dose. A comparison of the heat response for the two populations indicated that heating during early S phase induced thermotolerance for both cell killing and chromosomal aberrations; i.e., for 10% survival, which corresponded to 10% of the cells being cytologically normal, the thermal dose was twofold greater in the thermotolerant cells than in the control, nontolerant cells. Furthermore, this thermotolerance developed during S phase. These observations support the hypothesis that heating during S phase kills cells primarily by inducing chromosomal aberrations.     

Superoxide dismutase as an inhibitor of postischemic microvascular permeability increase in the hamster

The purpose was to elucidate the involvement of superoxide radical (O2-.) in the postischemic increase in the vascular permeability in the hamster cheek pouch. Cheek pouches of anesthetized hamsters were everted, prepared for intravital microscopy, and superfused with a bicarbonate buffered saline solution. Local ischemia for 30 min was obtained using a cuff placed around the proximal part of the cheek pouch. The vascular permeability in the postcapillary venules was quantified as leakage of intravenously injected fluorescein labeled dextran (FITC-dextran, Mw 150,000), using intravital microscopy and fluorimetry. There was a significant and reversible permeability increase after the reperfusion started. In the first series of experiments, combined intravenous infusion and topical application of human recombinant extracellular superoxide dismutase C (EC-SOD C) reduced the postischemic permeability response by 80%. Bovine CuZn-SOD given in exactly the same way reduced the response by 60%. In the second series of experiments, inactivated EC-SOD C was given to the control animals and active EC-SOD C was given to the treated animals. The topical treatment was excluded. Only active EC-SOD C reduced significantly the postischemic permeability increase when present during the ischemic period. Treatment with mannitol (i.v.) did not alter the postischemic response. Since active EC-SOD C and CuZn-SOD but not inactivated EC-SOD C effectively inhibited the response, we suggest that the superoxide anion is involved in the mediation of the postischemic permeability increase in the hamster.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.