Peritoneal Cavity Of Rats Research Articles

Ternatin, an Anti-Inflammatory Flavonoid, Inhibits Thioglycolate-Elicited Rat Peritoneal Neutrophil Accumulation and LPS-Activated Nitric Oxide Production in Murine Macrophages

Ternatin, an anti-inflammatory flavonoid from Egletes viscosa Less., was examined for its possible influence on thioglycolate-elicited neutrophil influx into the rat peritoneal cavity in vivo and nitric oxide production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages ex vivo. The neutrophil influx induced by thioglycolate was found to be significantly lower in ternatin (25 and 50 mg/kg, s. c.) pre-treated rats with a similar magnitude of inhibition produced by dexamethasone (1 mg/kg, s. c.), a known anti-inflammatory agent. Also, peritoneal macrophages from ternatin (25 mg/kg)-treated mice that were exposed to LPS demonstrated significantly less production of nitric oxide (NO). These results suggest that ternatin exerts its anti-inflammatory action, at least in part, through inhibition of neutrophil migration and modulation of macrophage function.

Induction of Angiogenesis in Omentum with Vascular Endothelial Growth Factor: Influence on the Viability of Encapsulated Rat Pancreatic Islets during Transplantation

Transplantation of pancreatic islets is proposed as a treatment for type 1 diabetes, but insufficient blood supply can cause the loss of viable grafted islets. In the present study, we investigated the influence of vascular endothelial growth factor (VEGF) on the angiogenesis of omentum during encapsulated islet allotransplantation and consequently on islet survival. Fifty rat islets, cultured for 24 h, were encapsulated in the presence or absence of human VEGF and implanted in the peritoneal cavity of rats (n = 6). After 7, 14 and 28 days of implantation, encapsulation devices with surrounding omentum were removed. Histological analysis of this tissue was performed. Cellular adhesion at the membrane surface was characterized by a phagocytosis test. The morphological aspect of the islets was analyzed and their functionality was evaluated by measuring insulin secretion. At each step of the study, there was a two-fold increase in the number of vessels in the presence of VEGF. In addition, VEGF increased the vessel diameter and the surface area of the angiogenic pedicle. Moreover, the presence of VEGF significantly decreased the distance between the devices and vessels (16.2 ± 5.6 vs. 51.6 ± 10.1 µm, p < 0.001). Membrane surface analysis showed a decrease in macrophage adhesion in the presence of VEGF. Furthermore, islet structure and functionality was preserved in the presence of VEGF. Stimulation of angiogenesis of omentum induced by VEGF is associated with preservation of islet viability. Local delivery of VEGF proved to be a relevant approach to ameliorate the outcome of islet transplantation.

Expression of a luxS gene is not required for Borrelia burgdorferi infection of mice via needle inoculation.

The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria. A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities. Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5 alpha, AI-2-like activity could not be detected within B. burgdorferi culture supernatants or concentrated cell lysates. Finally, a luxS-deficient mutant of B. burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation. Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B. burgdorferi.

Anti-inflammatory and smooth muscle relaxant activities of the hydroalcoholic extract and chemical constituents from Amburana cearensis A C Smith.

Amburana cearensis A. C. Smith, Fagaceae, is a medicinal plant commonly known as 'cumaru' and used in Northeast Brazil for the treatment of respiratory tract diseases. In the present work, we investigated the anti-inflammatory and smooth muscle relaxant activities of the hydroalcoholic extract (HAE), coumarin (Coum) and fl avonoid fraction (FF) isolated from the trunk barks of Amburana cearensis A. C. Smith. It was shown that HAE (200 and 400 mg/kg), Coum (20 and 40 mg/kg) and FF (40 mg/kg), administered orally, significantly inhibited both leukocyte and neutrophil migrations, in the carrageenan or N-formyl-methyl-leucyl-phenylalanine (fMLP)-induced migration in rat peritoneal cavity. The increase in cutaneous vascular permeability induced by serotonin in rats was significantly blocked by HAE (150 mg/kg, i.p.), Coum (5 mg/kg, i.p.) and FF (20 mg/kg, i.p.). However, only HAE blocked the histamine effect on Evans blue extravasation. In the guinea-pig trachea precontracted with carbachol (0.3 microM), histamine (0.1 microM) or KCl (0.1 M), the HAE, Coum and FF evoked a concentration-dependent relaxation in the presence of the three agonists. HAE (100-800 microg/ml) and Coum (4-32 microg/ml) also caused significant relaxation of the rat vas deferens previously contracted with adrenaline, acetylcholine or barium chloride. In addition, HAE, Coum and FF inhibited the histamine and serotonin-induced increase of cutaneous vascular permeability in rats.

Vascular permeabilization by intravenous arachidonate in the rat peritoneal cavity: antagonism by antioxidants

Arachidonic acid was investigated for its vascular permeabilizing potential in the rat peritoneal cavity and for its mechanism of action. The antagonistic potential of antioxidants (vitamin E, vitamin C and troxerutin) was also evaluated. Vascular permeability was equated to the rate of extravasation of Evans blue dye from plasma into the peritoneal cavity. Baseline permeability was linear up to 2 h, with a rate constant ( k) of 0.0031±0.0007 h −1. Intravenous arachidonate (from 30 μg/kg to 3 mg/kg) induced an immediate, dose-related and significant increase in permeability (ranging from 80% to 150%), which was comparable to the effect induced by similar doses of serotonin. Aspirin (10 mg/kg) reduced the arachidonate-induced permeability by 75%, but interestingly neither the stable thromboxane A 2 receptor agonist U46619 (prostaglandin H 2 endoperoxide epoxymethane) nor prostacyclin was able to increase peritoneal vascular permeability. In contrast, the permeabilizing action of arachidonic acid was very sensitive to antioxidant agents. Thus, vitamin C and the flavonoid compound troxerutin (100 mg/kg) fully abolished arachidonate-induced permeability, whereas vitamin E had only a partial effect (40–100% inhibition). In conclusion, intravenous administration of arachidonic acid strongly enhanced peritoneal vascular permeability in the rat, apparently via free radical generation. This rat peritoneal model can be used to evaluate the in vivo antinflammatory potential of antioxidant drugs.

Vascular permeabilization by intravenous arachidonate in the rat peritoneal cavity: antagonism by ethamsylate

The hemostatic agent, ethamsylate, inhibits arachidonic acid metabolism by a mechanism independent of cyclooxygenase activity and blocks carrageenan-induced rat paw edema. Here, ethamsylate was investigated for (i) in vivo actions on the free radical-dependent, permeabilizing responses to arachidonic acid and (ii) its antioxidant potential in vitro. Vascular permeability was equated to the extravasation rate of Evans blue from plasma into the rat peritoneal cavity. Antioxidant potential was investigated by classical in vitro tests for superoxide radicals, hydroxyl radicals (OH ·), and nitric oxide. Intravenous ethamsylate induced a very important and significant reduction of permeability responses to arachidonate, both when given preventively and cumulatively. Thus, (i) ethamsylate significantly reversed arachidonate-induced permeabilization, even at the lowest dose tested (44±5% at 10 mg/kg) and (ii) a maximal reversal (about 70%) was reached between 50 and 200 mg/kg ethamsylate. In contrast, ethamsylate (100 mg/kg) was unable to antagonize the vascular permeabilization induced by serotonin (5-HT). In antioxidant assays, ethamsylate showed scavenging properties against hydroxyl radicals generated by the Fenton reaction (H 2O 2/Fe 2+) even at 0.1 μM (−20±3%). OH · scavenging by ethamsylate reached 42±8% at 10 μM and 57±7% at 1 mM and was comparable to that of reference compounds (vitamin E, troxerutin, and mannitol). Conversely, ethamsylate was a poor scavenger of superoxide and nitric oxide radicals. In conclusion, intravenous ethamsylate potently antagonized the peritoneal vascular permeabilization induced by arachidonate, an action likely due to its antioxidant properties, particularly against hydroxyl radical. Such a mechanism can explain previous observations that ethamsylate inhibits carrageenan-induced rat paw edema. Whether it also participates in the hemostatic action of ethamsylate deserves further investigation.

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1.

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.

Porcine spermadhesin PSP-I/PSP-II stimulates macrophages to release a neutrophil chemotactic substance: modulation by mast cells.

The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) alpha antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFalpha by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.

Spermadhesin PSP-I/PSP-II heterodimer and its isolated subunits induced neutrophil migration into the peritoneal cavity of rats.

Spermadhesins are a group of (glyco)proteins from seminal fluid involved in various aspects of porcine fertilization. PSP-I/PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of porcine seminal fluid. Its biological function remains, however, enigmatic. Using an in vitro chemotaxis assay, we showed that PSP-I/PSP-II and its isolated subunits induced migration of purified neutrophils. A possible proinflammatory activity of PSP-I/PSP-II induced upon injection of the spermadhesin heterodimer and its isolated subunits into the peritoneal cavity of rats was investigated. Lavage of peritoneal cavities, thioglycolate treatment, and mast cell depletion were done before spermadhesin administration, and neutrophil migration was evaluated 4 h after injections. Pharmacological modulation was also investigated. Resident cell depletion by lavage reduced the neutrophil migration induced by PSP-I/PSP-II and the PSP-II subunit but had no effect on that induced by isolated PSP-I. Both an increase of macrophage population by thioglycolate treatment and mast cell depletion potentiated the neutrophil migration induced by PSP-I/PSP-II and by PSP-II. The glucocorticoid dexamethasone but not indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and BN50739 (platelet activation factor [PAF] antagonist) inhibited neutrophil migration induced by PSP-I/PSP-II. Coincubation with mannose-6-phosphate (a PSP-II-specific ligand) inhibited neutrophil recruitment induced by PSP-II but did not alter the PSP-I activity. As a whole, the data suggested that enhancement of the neutrophil migration-inducing activity of PSP-I/PSP-II and PSP-II involved an indirect mechanism, i.e., via activation of resident cells, probably macrophages. On the other hand, PSP-I appeared to act directly on neutrophils. We hypothesize that the neutrophil migration-inducing effect displayed by PSP-II might be due to interaction of its lectin domain with cellular receptors and that neutrophil recruitment induced by PSP-I may involve protein-protein interactions.

The influence of bovine casein-derived exorphins on mast cells in rodents

The secretagogue effects of α-casein (90–95) and β-casomorphin-7 on two populations of mast cells in rodents were investigated. Mast cells of “connective tissue type” were obtained from rat peritoneal cavity (PMC), and “mucosal-like type” mast cells (BMMC) were cultured from mouse bone marrow cells. β-Hexosaminidase activity was considered to be a marker of the cells’ secretory response. No reaction of BMMC on exorphins was found at up to millimolar concentrations. β-Casomorphin-7 and α-casein (90–95) exert secretagogue effect on PMC at milli- and micromolar concentrations, respectively. For this reason, further experiments were performed with α-casein (90–95) only. The PMC were activated in a non-cytotoxic, dose-, temperature-, and energy-dependent manner. Although PMC were responsive in a calcium and magnesium free buffer, the reaction was abolished when preincubation with EDTA was performed. Preincubation with opiate antagonist, naloxone, was effective only at millimolar concentration range. The significant decrease in PMC reactivity to α-casein (90–95) after preincubation with benzalkonium chloride suggests a peptidergic pathway of activation which involves a membrane-assisted but receptor-independent stimulation of G i-like proteins.

Tissue responses against immunoisolating alginate-PLL capsules in the immediate posttransplant period.

Alginate-polylysine (PLL) capsules are commonly applied for immunoisolation of living cells for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules with failure of the grafts as a consequence. Most studies addressing biocompatibility issues of alginate-PLL capsules have focused on the degree of overgrowth on the capsules after graft failure and not on the reaction against the capsules in the immediate posttransplant period. Therefore, capsules were implanted in the peritoneal cavity of rats and retrieved 1, 5, and 7 days later for histological examination and X-ray photoelectron spectroscopy analysis for evaluation of chemical changes at the capsule surface. After implantation, the nitrogen signal increased from 5% on day 0, to 8.6% on day 7, illustrating protein adsorption on the capsule's surface. This increase in protein content of the membrane was accompanied by an increase in the percentage of overgrown capsules from 0.5 +/- 0.3% on day 1 to 3.3 +/- 1.6% on day 7. The cellular overgrowth was composed of monocytes/macrophages, granulocytes, fibroblasts, erythrocytes, multinucleated giant cells, and basophils. This overgrowth was not statical as generally assumed but rather dynamic as illustrated by our observation that at day 1 after implantation we mainly found monocytes/macrophages and granulocytes that on later time points were substituted by fibroblasts. As the inflammatory reaction predictably interfere with survival of encapsulated cells, efforts should be made to suppress activities or recruitment of inflammatory cells. These efforts may be temporary rather than permanent because most inflammatory cells have disappeared after 2 weeks of implantation.

Sephadex induces eosinophil migration to the rat and mouse peritoneal cavity: involvement of mast cells, LTB4, TNF-alpha, IL-8 and PAF.

In this study we investigated the chemotactic mediators involved in the Sephadex-induced eosinophil migration into the peritoneal cavities of rats and mice, and which resident peritoneal cells release these mediators. Sephadex suspension was injected into the peritoneal cavities of rats or mice which were pretreated, or not, with specific drugs that inhibit synthesis or production of the inflammatory mediators and eosinophil chemotactic activities were observed. To investigate the role of resident peritoneal cells as a source of these chemotactic factors, the macrophage population was enhanced or the mast cell population was depleted. The resident cells were also stimulated, in vitro, with Sephadex and the chemotactic activity of the supernatants was determined. Sephadex induced dose and time dependent eosinophil migration in rats and mouse, which were inhibited by dexamethasone and MK 886. BN 52021 only affected the eosinophil migration into the mouse peritoneal cavity. An increase in the macrophage population did not alter the eosinophil migration induced by Sephadex in rat or mouse. However, mast cell population depletion reduced eosinophil migration in rats, but did not alter the migration in mice. Sephadex-stimulated rat mast cells released an eosinophil chemotactic factor whose release was inhibited by dexamethasone and MK 886. Anti-TNF-alpha and anti-IL-8 Abs inhibited the chemotactic activity of the mast cell supernatant. Sephadex-induced eosinophil migration into the rat peritoneal cavity is dependent on mast cells, which release LTB4, TNF-alpha and CINC-1. Conversely, Sephadex-induced eosinophil migration into the mouse peritoneal cavity is mediated by PAF and LTB4, which are not released from resident macrophages or mast cells.

Morphological Analysis of Iodoacetic Acid-Induced Cataract in the Rat

Purpose: To clarify the mechanism underlying the development of cataract in the rat lens after intraperitoneal administration of iodoacetic acid (IAA). Methods: (1) The 2% IAA dissolved in saline solution was injected at a dose of 40 mg/kg body weight into the rat peritoneal cavity. The retina and lens were intermittently extirpated and were examined by light and electron microscopy. (2) Two kinds of tracer, Evans blue (EB) and horseradish peroxidase (HRP), were injected into the tail veins and anterior chamber, and were observed with dissecting and electron microscopes. Results: (1) Four weeks after administration, a part of the lens epithelium at the lateral side of the lens was degenerated, and the lens nucleus developed faint turbidity after 8 weeks. After 16 weeks, the nuclear turbidity could not be observed because mild cortical opacity was developing. The epithelial degeneration recovered from around 12 weeks, and instead of spherical nuclei, elliptical nuclei appeared. (2) The EB dye injected into the tail vein significantly stained the ciliary body, where the anterior and posterior ciliary arteries anastomosed. EB injected from the lateral side of the lens was seen to move towards the lens nucleus. Electron microscopically, the epithelial degeneration of the ciliary body was observed. The incorporated HRP substance was found in the cytoplasm of the nonpigmented cells of the ciliary epithelium at an early stage after IAA administration. Conclusion: IAA injected intravenously first developed epithelial degeneration at the lateral side of the lens. This change induced swelling of the lens fibers in the lens nucleus. Recovered epithelial cells had a transformed nucleus, and in turn the cortical cataract was induced by a differentiation disorder of the lens fibers. These results indicate that the breakdown of the blood-aqueous barrier in the nonpigmented epithelium of the ciliary body is a trigger to cause the cataract. The IAA-induced cataract may be useful as an animal model of human age-related cataract.

Sulfite is generated from PAPS by activated neutrophils.

We previously reported that neutrophils produce sulfite in response to stimulation with lipopolysaccharide, and sulfite production is dependent on inorganic sulfate contained in culture media. Microorganisms such as yeast assimilate sulfate, during which process sulfite is generated by reduction of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an activated sulfate donor. However, little is known about how sulfite is produced in mammalian cells. In the current study, we demonstrated that chlorate, a specific inhibitor for PAPS synthesis, significantly suppressed production of sulfite by activated neutrophils obtained from rat peritoneal cavity that had been injected with glycogen to induce inflammation. Addition of excess amounts of PAPS could partially overcome the inhibitory effect of chlorate. Moreover, sulfite production from PAPS was clearly demonstrated in the cytosolic fraction of activated neutrophils. These findings strongly suggest that sulfite is generated, at least in part, from PAPS by activated neutrophils.

Role of nitric oxide on in vitro human eosinophil migration

Eosinophils purified from the rat peritoneal cavity have been found to contain nitric oxide synthase (NOS) functionally coupled to a cyclic GMP transduction pathway that is involved in in vitro eosinophil migration, but no studies on cell locomotion have been done with purified human eosinophils. Therefore, this study was carried out to investigate the effects of N ω -nitro- l-arginine methyl ester ( l-NAME; a non-selective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a type I/type II NOS inhibitor), 2-amino-5,6-dihydro-6-methyl-4 H-1,3-thiazine (AMT; a selective type II NOS inhibitor), and 1 H-[1,2,4]-oxidiazolo[4,3- a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor) on human eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Human eosinophils were purified from peripheral blood of healthy volunteers using a Percoll gradient followed by an immunomagnetic cell separator. Chemotaxis was evaluated using a 48-well microchemotaxis chamber. The fMLP (1.0 × 10 −7 M)-induced eosinophil migration was reduced significantly by l-NAME (0.1 and 1.0 mM), whereas the inactive enantiomer N ω-nitro- d-arginine methyl ester ( d-NAME) had no effect. The inhibition by l-NAME was restored by sodium nitroprusside (0.25 mM). The NOS inhibitors AMT and TRIM (0.05 to 0.25 mM each) also markedly attenuated fMLP-induced chemotaxis. Additionally, ODQ (0.01 to 0.5 mM) concentration-dependently inhibited fMLP-induced migration, and the inhibition was restored by 2.0 mM dibutyryl cyclic GMP. In conclusion, this study demonstrates that human eosinophils present a nitric oxide-cyclic GMP pathway that is involved in the in vitro locomotion of this cell type.

Nitric Oxide- and Oxygen-Derived Free Radical Generation from Control and Lipopolysaccharide-Treated Rat Polymorphonuclear Leukocyte

Previous studies from this lab have shown NO-mediated modulation of free radical generation from polymorphonuclear leukocytes (PMNs), following hypoxic-reoxygenation as well as in the normoxic cells. The present study is an attempt to investigate further the regulation of NO and free radical generation in the lipopolysaccharide (LPS)-treated PMNs. PMNs were isolated from the rat blood and peritoneal cavity, 4 h after LPS (1 mg/kg, i.p.) treatment. Nitric oxide synthase (NOS) activity and nitrite content were increased in the peripheral and peritoneal PMNs following LPS treatment. An increase in the apparent Vmax for l-arginine uptake was also observed in the LPS-treated peripheral PMNs, while peritoneal PMNs exhibited increase in both apparent Vmax and affinity for l-arginine. Synthesis of nitrite did not augment after increasing the availability of substrate to control PMNs, however, peripheral and peritoneal PMNs from LPS-treated rats utilized l-arginine more efficiently for nitrite synthesis. NOS activity, l-arginine uptake, and its utilization were maximal in the peritoneal PMNs. Arachidonic acid (AA, 1 × 10−6 M)-induced free radical generation from PMNs was also enhanced significantly after LPS treatment. Preincubation of PMNs with nitrite elevated the free radical generation and myeloperoxidase (MPO) release. MPO and antioxidant enzyme activity in the PMNs was significantly augmented after LPS treatment. NOS inhibitors, aminoguanidine and 7-nitroindazole, inhibited arachidonic acid-induced free radical generation from LPS treated PMNs. The results obtained thus indicate that augmentation of free radical generation from rat PMNs following LPS treatment appears to be regulated by NO and MPO.

Antioxidant properties of calcium dobesilate in ischemic/reperfused diabetic rat retina

Calcium dobesilate possesses antioxidant properties and protects against capillary permeability by reactive oxygen species in the rat peritoneal cavity, but whether a similar action can take place in the diabetic rat retina is unknown. We investigated the oral treatment of diabetic rats with calcium dobesilate on the prevention of free radical-mediated retinal injury induced by ischemia/reperfusion (90 min ischemia followed by 3 min and/or 24 h of reperfusion). Streptozotocin-induced diabetic rats were orally treated with 50 and 100 mg/kg of calcium dobesilate for 10 days ( n=12 in each group). In the first series of studies, calcium dobesilate was found to significantly reduce the maldistribution of ion content in diabetic ischemic/reperfused rat retina. Thus, in diabetic rats treated with 100 mg/kg/day calcium dobesilate, ischemia/reperfusion provoked: (i) 27.5% increase in retinal Na + content compared to 51.8% in the vehicle-treated group ( P<0.05), and (ii) 59.6% increase in retinal Ca 2+ content compared to 107.1% in vehicle-treated animals ( P<0.05). In the second series of studies, calcium dobesilate was found to significantly protect diabetic rat retina against inhibition of Na +/K +-ATPase and Ca 2+/Mg 2+-ATPase activities by ischemia/reperfusion (54% and 41% reduction, respectively, with 100 mg/kg of calcium dobesilate) and also against changes in retinal ATP, reduced glutathione (GSH), and oxidized glutathione (GSSG) contents. In the third series of experiments, rats treated with 100 mg/kg of calcium dobesilate reduced the hydroxyl radical signal intensity to 41% (measured by electron paramagnetic resonance), induced by ischemia/reperfusion in diabetic rat retina. Finally, 100 mg/kg calcium dobesilate significantly reduced retinal edema (measured by the thickness of the inner plexiform layer) in diabetic rats. In conclusion, oral treatment with calcium dobesilate significantly protected diabetic rat retina against oxidative stress induced by ischemia/reperfusion. Whether the antioxidant properties of calcium dobesilate explain, at least in part, its beneficial therapeutic effects in diabetic retinopathy deserves further investigation.

Regulation of OspE-related, OspF-related, and Elp lipoproteins of Borrelia burgdorferi strain 297 by mammalian host-specific signals.

In previous studies we have characterized the cp32/18 loci in Borrelia burgdorferi 297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37 degrees C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that B. burgdorferi-infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the -10 and -35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as B. burgdorferi is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease.

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.

Anti-inflammatory activity of cubebin, a lignan from the leaves of Zanthoxyllum naranjillo Griseb

Cubebin, a dibenzylbutyrolactone lignan isolated from the crude hexane extract of the leaves of Zanthoxyllum naranjillo, showed a significant anti-inflammatory activity by using the paw edema induced by carrageenin in rats, but did not provide a significant reduction in the cell migration for the acute carrageenin-induced inflammatory reaction in the peritoneal cavity of rats. Neither was it effective in reducing the edema induced by dextran nor the edema induced by histamine. It partially reduced the edema induced by serotonin. Moreover, it significantly reduced the edema induced by prostaglandin PGE 2 and the number of writhings induced by both acetic acid and PGI 2 in mice. Therefore, it may be suggested that the mechanism of action of cubebin is similar to that observed for most of the non-steroidal drugs.