Sucralfate attenuates gastric mucosal lesions and increased vascular permeability induced by ischaemia and reperfusion in rats.

Recent evidence suggests that oxygen-derived free radicals are involved in mediating gastric microvascular and parenchymal cell injuries induced by ischaemia and reperfusion. Therefore, the effect of the locally acting anti-ulcer drug, sucralfate, was studied on ischaemia and reperfusion (e.g. induced gastric lesions, intraluminal bleeding, changes in vascular permeability and non-protein sulfhydryl levels in the rat stomach). Allopurinol was used as a known standard antioxidant drug. Rats were subjected to 30 min of gastric ischaemia in the presence of 100 mmol/L hydrochloric acid and reperfusion periods of 15, 30 or 60 min duration. The gastric lesions were assessed microscopically under an inverted microscope. The vascular permeability was quantified by measuring the extravasated Evans blue in the stomach. There were significantly greater numbers of gastric lesions, intraluminal bleeding and leakage of Evans blue during all reperfusion periods as compared with those of ischaemia, with maximum effects occurring at 60 min following reperfusion. Pretreatment with sucralfate (31.25-250 mg/kg, p.o.) or allopurinol (12.5-50 mg/kg, i.p.) 30 min before the procedure, dose-dependently reduced the gastric lesions, intraluminal bleeding, and decreased the vascular permeability induced by ischaemia and reperfusion. Furthermore, sucralfate dose-dependently reverses the ischaemia and reperfusion-induced depletion of mucosal non-protein sulfhydryl levels and inhibited the superoxide radical production in both cell-free xanthine-xanthine oxidase and in the stimulated polymorphonuclear cellular systems. These results suggest that the protection produced by sucralfate against gastric injury may be due to its antioxidant effects.

The potential synergistic effect of calcium channel blockers and alpha-tocopherol on gastric mucosal injury induced by ischaemia-reperfusion.

METHODS AND RESULTS: Acute oral administration of various doses of alpha-tocopherol (100, 600, 1200 mg/kg by mouth) produced a dose dependent and significant protection of mucosal injury induced by ischaemia-reperfusion injury in experimental animals. In addition, intraperitoneal administration of calcium channel blockers, nifedipine (0.5, 1, 3 mg/kg), diltiazem (1.25, 2.5 mg/kg) and verapamil (1.25, 2.5, 5, 10 mg/kg) protected gastric mucosa in ischaemia-reperfusion. alpha-Tocopherol (100 mg/kg by mouth), when given in combination with either nifedipine (0.5 mg/kg intraperitoneally), diltiazem (1.25 mg/kg intraperitoneally) or verapamil (1.25 mg/kg intraperitoneally), significantly reduced gastric mucosal injury. The protective effect of calcium channel blockers and the antioxidant agent was synergistic. CONCLUSION: The results showed that calcium channel blockers potentiate the protective effect of the antioxidant activity of alpha-tocopherol on gastric mucosal injury induced by ischaemia-reperfusion in experimental animals.

The thermal potentiation of acetaminophen-inhibited PMN oxidative metabolism in vitro.

The effect of high temperatures (39, 41, and 43 degrees C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0-90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39-43 degrees C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O2-) production induced by AM was potentiated by incubating the cells at 39 or 43 degrees C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.

Role of oxygen-derived free radicals on gastric mucosal injury induced by ischemia-reperfusion.

A free radical is an unstable and highly-reactive chemical species capable of independent existence that contained one or more unpaired electrons in its outer orbital. A number of oxygen-derived free radicals (ODFRs) have been identified. However, superoxide (O(-)(2) and hydroxyl (OH*) radicals are extensively studied. The univalent reduction of oxygen to water produces a number of highly-reactive chemical intermediates such as O(-)2 and OH*, which are commonly-known as oxygen-derived free radicals. ODFRS may be formed from several sources as follows: a) mitochondrial cytochrome oxidase, b) xanthine oxidase, c) neutrophils and d) transitional metals. There are several important defense mechanisms to limit or to prevent the damage caused by excessive ODFRs activity. These antioxidant defenses can be divided into a) enzymatic defense mechanisms such as: superoxide dismutase (SOD): catalase: selenium-containing glutathione peroxidase and b) non-enzymatic defense mechanisms including: alpha-tocopherol; ascorbic acid; glutathione and any sulfhydryl-containing compounds.

Leishmania major: in vitro ultrastructural study of the paraxial rod of promastigotes.

The flagellum of Leishmania major promastigotes has an intraflagellar structure known as the paraxial rod (PAR) which extends from a point halfway in the flagellar pocket to the tip of the flagellum, lying opposite the axonemal microtubule doublets 4-7. An expansion of the axonemal plasma membrane envelops the PAR and may provide desmosomal attachment at the orifice of the flagellar pocket. The complex organization of the 4-6 nm thick filaments in the PAR was studied by us in cross, oblique, longitudinal and tangential sections by electron microscope. These filaments are disposed in two parallel lamellae, one alongside the axoneme (ca. 45 nm thick), and the other alongside the plasma membrane (ca. 65 nm thick), with an interlamellar gap of about 22-28 nm. In each lamella, 8-12 parallel series of longitudinal filaments at ca. 30 nm intervals interdigitate with coplanar parallel series of oblique filaments at ca. 25 nm intervals and inclined to the long axis of the flagellum at ca. 48 degrees, and ca. 55 degrees, in the inner (paraxonemal) and outer lamella, respectively. The parallel filaments in each of the longitudinal and oblique series are spaced at ca. 8 nm intervals. They are cross-striated at ca. 30 nm intervals by transverse filaments which terminate occasionally on adjacent axonemal microtubules 5 and 6 in the inner lamella, and the plasma membrane in the outer lamella. Extending across the interlamellar gap is a set of parallel rows of 7-12 nearly parallel filaments at ca. 20 nm intervals. The part of the flagellar plasma membrane enclosing the PAR has a subplasmalemmal cytoskeleton consisting of a layer of longitudinal 2 nm filaments at 8 nm intervals, obliquely striated by parallel 2 nm filament doubles at ca. (-65) degrees with the long axis of the flagellum and ca. 20 nm periodicity. Each filament doublet stria apparently gives origin to collinear short filament doublet extensions that curve into juxtaposed meshes of the outer lamella. Microtubules of the axonemal doublets 5 and 6 are connected to electron-dense (ca. 12 nm thick) strips of the inner lamella of the PAR by longitudinal series of ca. 4 nm cross-links across a ca. 12 nm cleft.

The pattern of respiratory burst of leucocytes in patients with Echinococcus granulosus.

The function of polymorphonuclear leucocytes (PMN) of patients with hydatidosis was investigated. The patients were divided into three categories according to the characteristics of the cyst (calcified, alive and dead cyst). Healthy blood donors were used as a control group. The oxidative activity of PMN was determined by chemiluminescence (CL) assay. Reduction of ferricytochrome C was used to measure the superoxide (O2-) production. Phagocytosis was monitored by opsonized yeast uptake. The results showed that the CL response, O2- production and phagocytic index of PMN, significantly increased in patients with dead cysts compared with healthy subjects while in patients with live cysts there was a marked reduction. No significant changes were noticed in patients with calcified cysts. These data indicate that the PMN of infected patients were in an activated state both functionally and metabolically.

Effect of Entamoeba histolytica toxins on isolated human polymorphonuclear leukocytes' phagocytic function.

This study was undertaken to investigate the effect of Entamoeba histolytica toxin (Ehp/t) obtained from HM1:IMSS strain on human polymorphonuclear leukocytes (PMNs) phagocytic function. A less virulent strain, i.e., NIH:200 was also used in this study for comparison. The results revealed that the toxin obtained from the strain HM1:IMSS inhibited the phagocytic activity of PMNs as measured by yeast uptake or chemiluminescence response while the toxin of strain NIH:200 did not show any significant effect. On the other hand, washing of PMNs that had been pre-exposed to the toxin of strain HM1:IMSS failed to reverse the effect of toxin on PMNs. Heat inactivation of the toxin failed to alter its effect on PMNs. Addition of N-acetyl-D-galactosamine (Ga1NAc) to the PMNs did not alter to activity of Ehp/t toxin. These fingdings suggested that Ehp/t toxin might induce similar effect on PMNs of patients infected with ameba.

Verapamil enhances the inhibitory effect of diclofenac on the chemiluminescence of human polymorphonuclear leukocytes and carrageenan-induced rat's paw oedema.

Diclofenac dose-dependently (1.25, 2.5 and 5 mg/kg, i.p.) inhibited the oedema produced by carrageenan in the rat's paw. This anti-inflammatory effect was enhanced by the co-administration of various doses (10, 20, and 30 mg/kg i.p.) of verapamil. Diclofenac or the calcium channel blocker, verapamil, when added separately, inhibited the chemiluminescence (CL) response of isolated human polymorphonuclear leukocytes (PMNs) stimulated by either the soluble agent, phorbol myristate acetate, (PMA) or by particulate opsonized zymosan (OPZ) in a dose-dependent manner. When verapamil was combined with diclofenac, in vitro, the inhibitory effect on PMA or OPZ-induced CL response was synergistic. This inhibitory effect on either diclofenac or verapamil on the isolated PMNs was readily reversible when the PMNs were washed with phosphate buffered saline (PBS). Additionally, diclofenac or verapamil did not significantly affect the viability of PMNs. It is concluded that verapamil enhances the anti-inflammatory effect of diclofenac in vivo and potentiates its inhibitory effect on the CL of isolated human PMNs in vitro.

Effect of Leishmania major on human polymorphonuclear leucocyte function in vitro.

The effect of various antigens of Leishmania major promastigotes on the function of human polymorphonuclear leucocytes (PMNLs) was examined. Different concentrations of L. major antigens were incubated with isolated PMNLs for various periods and the respiratory burst was assessed by Luminol-dependent chemiluminescence. All the Leishmania antigens employed inhibited the PMNL respiratory burst by 35-64%. PMNL viability was not affected either by the concentrations or type of the parasite antigen. Oxygen free radical scavengers enhanced the action of the antigens on the PMNL respiratory burst.

Hepatic microsomal protein and cytochrome P-450 in BALB/c mice infected with Leishmania.

The effects of infection of mice with Leishmania major on liver microsomal protein and cytochrome P-450 were examined. The levels of hepatic microsomal protein and cytochrome P-450 were monitored at 6, 7, 9 and 12 weeks post-infection. The results indicated that the amount of hepatic microsomal protein and cytochrome P-450 were unchanged throughout the course of infection with L. major, despite the high degree of parasite proliferation in Kupffer cells and marked reduction in phagocytosis. The current results clearly indicate that Leishmania-induced macrophage suppression has no inhibitory effect on hepatic microsomal protein and cytochrome P-450.

Effect of Leishmania major on luminol-dependent chemiluminescence of human whole blood phagocytosis.

The aim of this study is to examine the effect of L. major on human whole blood phagocytosis. The phagocytosis was activated by phorbol myristate acetate (PMA) or opsonized zymosan. Various types of Leishmania antigens were tested on luminol-dependent chemiluminescence. In PMA-activated whole blood phagocytosis group, promastigotes and infected medium significantly (P less than 0.001) depressed the maximum peak of chemiluminescence to 5.77 +/- 1.6 and 5.65 +/- 0.45 mV respectively, compared to 9.85 +/- 22 in control group. In a similar fashion a significant reduction of the area under the curve was noted. On the other hand, opsonized zymosan-activated phagocytosis was significantly (P less than 0.025) inhibited in the promastigote treated group only. The maximum peak response was 4.68 +/- 1.5 and 10.8 +/- 0.46 mV in promastigote and control groups, respectively. The above data indicated; a) an inhibition of whole blood phagocytosis induced by Leishmania and b) a marked reduction of the oxygen-reactive metabolites radicals generated by whole blood phagocyte cells and measured by luminol-dependent chemiluminescence suggesting the important role of oxygen free radicals in the pathogenesis of leishmaniasis.

Entamoeba histolytica depresses chemiluminescence in stimulated human polymorphonuclear leukocytes.

Effect of Entamoeba histolytica proteinase/toxin (Ehp/t) on the luminol-dependent chemiluminescence (CL) in stimulated human polymorphonuclear leukocytes (PMN) was studied. The role of superoxide (SO) and hydroxyl (OH) anions in the Ehp/t-associated enhancement/inhibition of CL was also studied using specific scavengers and a biological response modifier, muramyldipeptide (MDP). Ehp/t was isolated from axenic trophozoites of the HM-1:IMSS strain of virulent strain of E. histolytica. Proteinase activity was assayed on a synthetic substrate, Z-arg-arg-AFC and cytotoxicity was tested on HeLa cell monolayers. PMN isolated from blood were incubated with Ehp/t prior to stimulation by phorbol myristateacetate (PMA, 2 micrograms/ml), serum-treated zymosan (2.5 mg/ml) and glucan (2 mg/ml). CL was monitored in an LKB (Wallac) Luminometer. Ehp/t was found to depress up to 90% of CL induced by PMA, glucan and zymosan. Such a depression was Ehp/t concentration-dependent. A 150 micrograms/ml concentration of Ehp/t, obtained from a 0.015-1.5 mg/ml concentration range tested at different incubation times and temperatures, was used in most of our experiments. Incubation time and temperature optima were 15 min and 37 degrees C, respectively. Ehp/t partially inhibited the CL associated with SO and OH. MDP, in the presence of Ehp/t, enhanced CL response in human PMN to about 67% with reference to normal CL without inhibitor. PMN were confirmed to play a vital role in amebic tissue invasion mechanisms.

Effect of glucan on Leishmania major infection in BALB/c mice.

The effect of glucan on Leishmania major infection was studied in BALB/c mice, which are highly susceptible to leishmania infection. Glucan (0.45 mg), or isovolumetric dextrose, was administered intraperitoneally 7, 5, 3 and 1 day before infection with L. major promastigotes. At 3, 5, 6, 8 and 10 weeks after infection, animals were killed; the liver and spleen of each animal were weighed and the parasite burden was calculated. A significant (p less than 0.01) reduction in amastigote proliferation in liver and spleen of animals pretreated with glucan was demonstrated 4, 6 and 8 weeks after infection.