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.     

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 molecular role of mast cells in atherosclerotic cardiovascular disease

Human atherosclerosis has many characteristics of an inflammatory disorder. Recent data suggest that mast cells might be important in the pathogenesis of atherosclerotic disease. By secretion of pro-inflammatory cytokines, mast cells can assist in the recruitment of monocytes and lymphocytes into vascular tissue, thereby propagating the inflammatory response. Mast cell enzymes might activate pro-metalloproteinases, thereby destabilizing atheromatous plaques. Mast cells can facilitate foam cell formation by promoting cholesterol accumulation. However, mast cell tryptase could slow thrombus formation at sites of plaque rupture by interfering with coagulation. Therefore, mast cells can modulate coronary artery disease by both facilitatory and inhibitory pathways.     

Remodeling of neural, glial, and tracheal cell populations in the developing Manduca wing

In the rat larynx, plasma exudation and edema formation were studied by light and electron microscopy after i.v. injections of the mast cell activator compound 48/80, substance P, and capsaicin. The morphological effects of substance P and capsaicin on connective tissue mast cells in vivo were also examined. Of the drugs tested, only compound 48/80 degranulated the connective tissue mast cells. All drugs induced a subepithelial plasma exudation in the subglottic region, with edema in the lamina propria and widened intraepithelial intercellular spaces, though the tight junction regions seemed intact. In the epiglottis, 10 min after compound 48/80 injection, there was edema in the lamina propria on the lingual side, with an intact and tight epithelial lining. No morphological sign of edema was found in the epiglottis after injection of substance P or capsaicin. The pronounced effect found in the epiglottic region after compound 48/80 injection was due to the release of mediators such as histamine and 5-hydroxytryptamine from the connective tissue mast cells. This study supports the belief that substance P in vivo mediates an increased vascular permeability by a direct effect on the blood vessels – a mechanism distinct from mast cell degranulation.     

Mast cells mediate the microvascular inflammatory response to systemic hypoxia.

Systemic hypoxia produces an inflammatory response characterized by increases in reactive O(2) species (ROS), venular leukocyte-endothelial adherence and emigration, and vascular permeability. Inflammation is typically initiated by mediators released from activated perivascular cells that generate the chemotactic gradient responsible for extravascular leukocyte accumulation. These experiments were directed to study the possible participation of mast cells in hypoxia-induced microvascular inflammation. Mast cell degranulation, ROS levels, leukocyte adherence and emigration, and vascular permeability were studied in the mesenteric microcirculation by using intravital microscopy of anesthetized rats. The main findings were 1) activation of mast cells with compound 48/80 in normoxia produced microvascular effects similar, but not identical, to those of hypoxia; 2) systemic hypoxia resulted in rapid mast cell degranulation; 3) blockade of mast cell degranulation with cromolyn prevented or attenuated the hypoxia-induced increases in ROS, leukocyte adherence/emigration, and vascular permeability; and 4) mast cell degranulation during hypoxia was prevented by administration of the antioxidant lipoic acid and of nitric oxide. These results show that mast cells play a key role in hypoxia-induced inflammation and suggest that alterations in the ROS-nitric oxide balance may be involved in mast cell activation during hypoxia.     

Neurogenic inflammation, vascular permeability, and mast cells. II. Additional evidence indicating that mast cells are not involved in neurogenic inflammation.

Activation of cutaneous sensory nerves induces vasodilatation and vascular permeability, i.e., neurogenic inflammation. We examined the histology and possible mast cell involvement in cutaneous neurogenic inflammation induced by electrical nerve stimulation (ENS). Three lines of evidence indicated that mast cells were not involved in rodent cutaneous neurogenic inflammation induced by electrical stimulation of the saphenous nerve. 1) Most mast cells (86.5% of all mast cells in the dorsal skin of the paw) were found in the deep dermis, whereas vessels developing increased vascular permeability after nerve stimulation (visualized with the supravital dye Monastral blue B, a macro-molecular tracer) were localized predominantly in the superficial dermis. By contrast, i.v. substance P, which also causes increased cutaneous vascular permeability, predominantly caused deeper vessels to leak. As analyzed by electron microscopy, the vessels that developed permeability in response to nerve stimulation, and were thereby stained with Monastral blue B, were found to be exclusively postcapillary venules. 2) Disodium cromoglycate (DSCG), a mast cell stabilizing compound, inhibited the cutaneous vascular permeability induced by intradermal injections of anti-IgE in a dose-dependent manner. By contrast, vascular permeability induced by ENS was not influenced by disodium cromoglycate treatment. 3) ENS and i.v. substance P both induced cutaneous vascular permeability in mast cell-deficient W/Wv mice, despite the fact that their skin contained only 4.7% of the mast cells present in their normal +/+ litter mates. The magnitude of ENS-induced vascular permeability responses in W/Wv mice were similar to control +/+ and BALB/c mice. This study supports our earlier observations suggesting that mast cell activation is not essential for the initial, vascular permeability phase of neurogenic inflammation in rodent skin.     

The monocyte-macrophage-mast cell axis in dengue pathogenesis

Dengue virus, the causative agent of dengue disease which may have hemorrhagic complications, poses a global health threat. Among the numerous target cells for dengue virus in humans are monocytes, macrophages and mast cells which are important regulators of vascular integrity and which undergo dramatic cellular responses after infection by dengue virus. The strategic locations of these three cell types, inside blood vessels (monocytes) or outside blood vessels (macrophages and mast cells) allow them to respond to dengue virus infection with the production of both intracellular and secretory factors which affect virus replication, vascular permeability and/or leukocyte extravasation. Moreover, the expression of Fc receptors on the surface of monocytes, macrophages and mast cells makes them important target cells for antibody-enhanced dengue virus infection which is a major risk factor for severe dengue disease, involving hemorrhage. Collectively, these features of monocytes, macrophages and mast cells contribute to both beneficial and harmful responses of importance to understanding and controlling dengue infection and disease.     

The role of mast cells in vascular permeability disorders in rats subjected to immobilization stress]

Disturbances of vascular permeability were studied by the "vascular labeling" technique in the mesentry during the 24-hour immobilization of rats. Administration of dimebolin (an antihistaminic preparation) decreased the number of labeled vessels and labeling intensity. This effect was expressed in the presence of mast cells only and was accompanied by the mast cell degranulation. The authors suppose that the mast cells contain a substance preventing the disturbance of vascular permeability and released during degranulation. Such substance might be heparin. Experiments showed that small doses of heparin failed to produce such effect. These results allowed one to conclude that mast cells played a double role in the mechanisms of disturbance of vascular permeability during immobilization--the damaging (by the action of histamine and serotonine) and the protective (by the released heparin) action.     

Influence of tilapia mast cell lysate on vascular permeability

Kinetics of vascular permeability was determined by measuring the amount of Evans blue leaked into the tilapia mast cell (tMC)-lysate injection site. Injection with tMC lysate enhanced the vascular permeability. The response consisted of three distinct phases, the first started immediately after the injection, the second started at about 2 h, reaching its maximum at 4 h, and the third response started at 12 h and continued for more than 24 h. Heating of the tMC lysate at 100 degrees C for 10 min had no effect on the first response, while the second response was significantly reduced by heating at 50 degrees C for 10 min. The tMCs seem to have two kinds of factors that enhance vascular permeability. The tMC lysate induced Ca2+ uptake by cultured tilapia endothelial cells, indicating that tMC products directly activate the endothelial cells and increase vascular permeability similar to products of mammalian mast cells. These results indicate that with respect to influence on vascular permeability tilapia mast cells resemble the mast cells of mammals.     

Temperature evolution in tissues embedded with large blood vessels during photo-thermal heating

During laser-assisted photo-thermal therapy, the temperature of the heated tissue region must rise to the therapeutic value (e.g., 43 °C) for complete ablation of the target cells. Large blood vessels (larger than 500 micron in diameter) at or near the irradiated tissues have a considerable impact on the transient temperature distribution in the tissue. In this study, the cooling effects of large blood vessels on temperature distribution in tissues during laser irradiation are predicted using finite element based simulation. A uniform flow is assumed at the entrance and three-dimensional conjugate heat transfer equations in the tissue region and the blood region are simultaneously solved for different vascular models. A volumetric heat source term based on Beer–Lambert law is introduced into the energy equation to account for laser heating. The heating pattern is taken to depend on the absorption and scattering coefficients of the tissue medium. Experiments are also conducted on tissue mimics in the presence and absence of simulated blood vessels to validate the numerical model. The coupled heat transfer between thermally significant blood vessels and their surrounding tissue for three different tissue-vascular networks are analyzed keeping the laser irradiation constant. A surface temperature map is obtained for different vascular models and for the bare tissue (without blood vessels). The transient temperature distribution is seen to differ according to the nature of the vascular network, blood vessel size, flow rate, laser spot size, laser power and tissue blood perfusion rate. The simulations suggest that the blood flow through large blood vessels in the vicinity of the photothermally heated tissue can lead to inefficient heating of the target.     

UVA-induced calcium oscillations in rat mast cells

UVA is a major bio-active component in solar irradiation, and is shown to have immunomodulatory and anti-inflammatory effects. The detailed molecular mechanism of UVA action in regard to calcium signaling in mast cells, however, is not fully understood. In this study, it was found that UVA induced ROS formation and cytosolic calcium oscillations in individual rat mast cells. Exogenously added H2O2 and hypoxanthine/xanthine oxidase (HX/XOD) mimicked UVA effects on cytosolic calcium increases. Regular calcium oscillation induced by UVA irradiation was inhibited completely by the phosphatidylinositol-specific phospholipase C inhibitor U73122, but U73343 was without effect. Tetrandrine, a calcium entry blocker, or calcium-free buffer abolished UVA-induced calcium oscillations. L-type calcium channel blocker nifedipine and stores-operated calcium channel blocker SK&F96365 had no such inhibitory effect. ROS induction by UVA was abolished after pre-incubation with anti-oxidant NAC or with NAD(P)H oxidase inhibitor DPI; such treatment also made UVA-induced calcium oscillation to disappear. UVA irradiation did not increase mast cell diameter, but it made mast cell structure more granular. Spectral confocal imaging revealed that the emission spectrum of the endogenous fluorophore in single mast cell contained a sizable peak which corresponded to that of NAD(P)H. Taken together, these data suggest that UVA in rat mast cells could activate NAD(P)H oxidase, to produce ROS, which in turn activates phospholipase C signaling, to trigger regular cytosolic calcium oscillation.     

Interactions of mast cells and leukocytes in increased vascular permeability in the focus of inflammation]

On the model of acute infectious peritonitis in rats it is shown that the mast cell depletion affected the inflammatory focus vascular permeability mainly in the immediate phase of its increase. Leukopenia inhibited the permeability both in the immediate and delayed phases. The combined depletion of mast cells and leukocytes not only inhibited the degree of vascular permeability increase but strongly affected its kinetics during exudative phase of peritonitis. The results indicate that in the natural conditions of inflammation the mast cell-leukocyte interaction in the vascular permeability increase takes place.     

Collagen concentration and mast cell in perinatal murine uterus

Edema was found in the uterine tissue on the day of parturition. The number of mast cells was increased significantly on this day, indicating that the time of appearance of the edema is identical to the time of a significant increase in the number of mast cells. These results suggest that mast cells induce the high vascular permeability that permits passage of collagenase-activating proteases to the uterine tissue from the serum.     

Modeling the effect of blood vessel bifurcation ratio on occlusive thrombus formation

Vascular geometry is a major determinant of the hemodynamics that promote or prevent unnecessary vessel occlusion from thrombus formation. Bifurcations in the vascular geometry are repeating structures that introduce flow separation between parent and daughter vessels. We modelled the blood flow and shear rate in a bifurcation during thrombus formation and show that blood vessel bifurcation ratios determine the maximum shear rate on the surface of a growing thrombus. We built an analytical model that may aid in predicting microvascular bifurcation ratios that are prone to occlusive thrombus formation. We also observed that bifurcation ratios that adhere to Murray’s law of bifurcations may be protected from occlusive thrombus formation. These results may be useful in the rational design of diagnostic microfluidic devices and microfluidic blood oxygenators.     

Serotonin metabolism in thrombocytes and microvascular hemostasis in spontaneous arterial hypertension

Serotonin content and accumulation in platelets and its release from them, as well as changes in thrombus formation in mesenteric arterioles and venules of the small intestine have been investigated in control rats and rats with spontaneous hypertension (SHR). Serotonin accumulation in platelets was determined upon its incubation with platelets. Disodium ADP salt was used as an inductor of release. Laser-induced thrombosis was caused by microvessels exposure to impulse laser irradiation. The control animals revealed a significant difference between the initial serotonin platelet level and serotonin level upon incubation and release; in values, the values of basic thrombus-forming parameters were higher than in arterioles. In SHR there is a decrease in biogenic amine content in platelets, a depression in its accumulation and release, an increase in the time of thrombus growth, its size up to the separation of the first embolus and its length along the vascular wall. It is concluded that spontaneous hypertension is characterized by decreased functional activity of platelets and depressed resistance of arterioles and venules to thrombus formation.     

Studies in experimental animal models of the effect of ultraviolet radiation (UVC) on blood and isolated cell populations

The cellular and molecular basis of the therapeutically used effect of reinjected ultraviolet ( UVC ) irradiated blood is unknown. First approaches to that problem were made in this study by aid of model experiments. Neither the spontaneous degranulation nor the antigen-induced histamine release from rat connective tissue mast cells (in vivo) was influenced by the injection (i.v.) of ultraviolet irradiated blood or blood lymphocytes. By comparison of the effect of ultraviolet light on blood lymphocytes (number of dead cells, strength of chemoluminescence) after irradiation of the isolated cells and the unfractionated blood respectively, we could show that the strong light absorption within the blood sample prevents damage or functional alterations of the blood lymphocytes. The compound 48/80-induced histamine release from rat peritoneal mast cells can be completely inhibited by ultraviolet irradiation (0.6 mJ/cm2) without increasing the spontaneous histamine release.     

Role of the protease in the permeability enhancement by Vibrio vulnificus

The protease produced by Vibrio vulnificus enhances vascular permeability through histamine release from mast cells and activation of the plasma kallikrein-kinin system which generates bradykinin when injected into the dorsal skin. V. vulnificus living cells also enhanced vascular permeability within a few hours after the injection into the dorsal skin. The permeability-enhancing activity of living cells was greatly reduced by addition of soybean trypsin inhibitor, a specific inhibitor for plasma kallikrein-kinin system, or anti-protease IgG. Two protease-deficient mutants induced by nitrosoguanidine treatment had only one-tenth permeability-enhancing activity of a wild-type strain. These results indicate that V. vulnificus elaborates the protease in vivo and that the protease elaborated enhances vascular permeability through release of chemical mediators such as histamine and bradykinin and forms edema.     

低能量激光与集落刺激因子对人脐带血造血细胞的增殖作用

本研究用铜蒸气激光照射人脐带带血造血细胞,观察低能量激光与集落因子对造血细胞的增殖作用。结果显示激光加ＣＳＦ对造血细胞ＧＭ－ＣＦＵｃ有协同增殖作用,与单用激光照射组,ＣＳＦ刺激组及对照组相比,有非常显著性差异。     

Mast cells in the focus of an acute infectious inflammation]

On the model of E. coli-induced acute infectious peritonitis in rats it is established that the mast cell reaction and histamine level increase in exudate and inflamed mesentery tissue are biphase and are observed predominantly following the inflammatory agent action, in the period corresponding to the immediate phase of peritoneal cavity vessel permeability increases. The preliminary elimination of mast cells significantly inhibits a rise in the vascular permeability in the immediate phase and slightly affects the delayed phase, thus prolonging exudation. At the same time the dynamics of free histamine indicates its direct involvement in mediation and/or modulation as well as in subsequent inflammatory events. The common rules of mast cell involvement and vascular permeability increase in infectious and aseptic inflammation have been shown.