The polysaccharide from Tamarindus indica (TS-polysaccharide) protects cultured corneal-derived cells (SIRC cells) from ultraviolet rays.

The aim of this work was to investigate the possible protective effect of a new viscosising agent, TS-polysaccharide, on corneal-derived cells (SIRC) exposed to ultraviolet-B rays. To verify this, SIRC cells were first exposed, in the absence or in the presence of TS-polysaccharide (1% w/v), for 9 s at the UV-B source and then post-incubated for 45 min at 37 degrees C. After this period the hydrogen peroxide (H(2)O(2)) accumulated in the medium and the concentration of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) in cell DNA was measured. In addition, the amount of (3)H-methyl-thymidine incorporated in cellular DNA was evaluated after 18 h from irradiation. Our results show that cells exposed to UV-B rays accumulate H(2)O(2), and have higher levels of 8OHdG and a lower amount of (3)H-methyl-thymidine incorporated in DNA than control cells. In the presence of TS-polysaccharide, the H(2)O(2) and 8-OHdG accumulation, and the (3)H-methyl-thymidine incorporation were significantly reduced with respect to the values measured in cells exposed in the absence of the polysaccharide. We propose a protective role of the polysaccharide in reducing UV-B derived DNA damage to eye cells. This finding could be of some clinical importance when the polysaccharide is used as a delivery system for ophthalmic preparations.

Pharmacokinetics of oxatomide in preterm infants.

The pharmacokinetics and tolerability of oxatomide oral suspension were investigated in preterm infants to evaluate the feasibility of planning a further study to assess its antiinflammatory effects and its effectiveness in preventing chronic lung disease (CLD). Following the administration of oxatomide 1 mg/kg, the peak plasma concentration (Cmax), the elimination half-life (t1/2), the volume of distribution (Vd), and the area under the curve (AUC) 0-36 h were measured and the following results were obtained: 42.2 +/- 15 ng/ml at 2 h after oxatomide administration, 41.4 +/- 2.0 h, 37.4 +/- 4.2 l/kg, and 468 +/- 52 ng/ml/h, respectively. Our study, therefore, demonstrated that a dose of 1 mg/kg/day oxatomide was effective in reaching therapeutic plasma levels in preterm infants without inducing adverse effects.

Ovalbumin sensitization of guinea-pigs reduces fMLP-induced calcium signal in alveolar macrophages.

In this study we analyzed the N-formyl-Met-Leu-Phe (fMLP)-induced calcium signal in alveolar macrophages (AM) isolated from ovalbumin-sensitized (OA-sensitized AM) and naive (naive AM) guinea-pigs. Guinea-pigs were sensitized by subcutaneous injection of OA and AM were isolated by bronchoalveolar lavage 6 weeks thereafter. On the following day, we measured in resting and fMLP-stimulated cells: intracellular calcium concentration by fura-2 imaging analysis, forskolin-induced cyclic AMP production and superoxide dismutase inhibitable superoxide anion release of adherent AM. Resting calcium was 82+/-5.0 nM (n=217) and 144+/-9.3 nM (n=213, P<0.001) in naive and OA-sensitized AM respectively. fMLP (10(-11)-10(-7)M) induced a dose-dependent calcium increase, 10(-8)M being the maximal effective dose in both naive and OA-sensitized AM. However, at all doses tested, this fMLP effect was lower in OA-sensitized than in naive AM. While in resting condition 10(-5)M forskolin increased cyclic AMP both in naive and OA-sensitized AM, in fMPL-stimulated AM forskolin was effective only in OA-sensitized AM. Superoxide anion release measured 10 min after fMLP stimulus was higher in naive than in sensitized AM. These data suggest that the fMLP-induced intracellular signal is different in OA-sensitized AM compared to naive cells.

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Mechanical stretch reveals different components of endothelial-mediated vascular tone in rat aortic strips.

1. Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. 2. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium-preserved strips were precontracted with either phenylephrine or prostaglandin F(2 alpha) (PGF(2 alpha)). 3. In control conditions, acetylcholine (ACh) dose-dependently relaxed phenylephrine-precontracted strips independently of resting tension. 4. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh-induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose-response curve to the right. 5. After preincubation with indomethacin (5 microM) or ibuprofen (10 and 100 microM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium-dependent, dose-dependent contraction. ACh (10(-6) M) increased the contraction up to two times greater the phenylephrine-induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF(2 alpha). 6. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX-derived metabolites, can relax precontracted strips. COX inhibition up-regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase-dependent, ACh-induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh-induced relaxation marginally.

Protective effect of pirenoxine and U74389F on induced lipid peroxidation in mammalian lenses. An in vitro, ex vivo and in vivo study.

Oxidative stress is believed to be involved in cataract development. The protective effect of the xanthomatine derivative, pirenoxine, and the 21-aminosteroid U74389F on oxidative insult in mammalian lenses was evaluated in vitro, ex vivo and in vivo. In vitro pirenoxine and U74389F inhibited lipid peroxidation induced by iron or haemoglobin in guinea-pig homogenate lens or whole lenses. Both compounds produced the same effect when lens oxidation was induced by superoxide producing system such as xanthine/xanthine oxidase or fMLP stimulated macrophages. In all the in vitro experiments, the values of biochemical lipid peroxidation markers, such as lipid hydroperoxides or thiobarbituric reactant substances, fell to the basal values with the addition of either pirenoxine (10(-5) M) or U74389F (10(-5) M). When two drops (60 microl) of the above molecular solutions (0.005 and 0.012% in saline respectively) were instilled in rabbit eyes (every hour for 8 hours over 2 days), the extracted lenses appeared to have better defences against an in vitro iron-induced lipid peroxidation, as shown by the values of conjugated dienes and lipid soluble fluorescent substances. These values also proved to be significantly lower when the same parameters were assayed in lenses from eyes where a lipid peroxidation was induced in vivo by haemoglobin or Diquat intravitreal injection followed by instillations of pirenoxine sodium salt or U74389F solutions (2 drops of about 60 microl every hour for 8 hours over 4 days) administered topically. Polarographic and chronocoulometric measurements were also performed in order to investigate the action mechanisms of both compounds. Experimental data indicate that the pirenoxine sodium salt and U74389F may be considered effective tools for rejecting an oxidative attack on the lenses, which can finally lead to cataract formation.

An in vivo, ex vivo and in vitro comparative study of activity of copper oligopeptide complexes vs Cu(II) ions.

The tetrapeptide-Cu(II) complex H-(l-His-Gly)2-OH/Cu(II), indicated as L-Cu(II), has been investigated, as compared to the Cu(II) inorganic salt CuSO4, for its antioxidative and anti-inflammatory properties under a panel of experimental conditions. Both inorganic and organic Cu(II) compounds showed comparable activities in vitro and ex vivo by: (i) protecting, in a dose-dependent manner, rat brain homogenates from Fe(III)/ascorbate- or haemoglobin-induced lipid peroxidation; (ii) inhibiting the superoxide-mediated ferricytochrome c reduction by activated macrophages. CuSO4 and L-Cu(II) also exhibited similar anti-inflammatory effects in vivo by reducing significantly the extent of carrageenan-induced edema in the rat paw. The activities of the two compounds diverged strikingly only in the xanthine/xanthine oxidase system at low phosphate buffer concentration. L-Cu(II) decreased the rate of NBT reduction by superoxide in a true SOD-like fashion without affecting urate production. Instead, Cu(II) ions caused the rapid xanthine oxidase inactivation thus inhibiting both urate and superoxide production; this effect might be ascribed to the superoxide-mediated generation of the strong oxidant Cu(III) and its interaction with the enzyme. The administration of Cu(II), whether complexed with linear oligopeptides or as an inorganic salt, to animals or tissue extracts, conferred protection against oxidation and ought, conceivably, to interact with endogenous biological molecules and form highly bioavailable complexes which serve, subsequently, as the real scavengers. Moreover, the claimed prominent scavenger activities of Cu(II)-oligopeptide complexes over inorganic copper ions could be realised only in very simple in vitro systems through mechanisms which, although of biochemical interest, are unlikely to be of physiopathological significance.

Influence of interleukin 1alpha on superoxide anion, platelet activating factor release and phospholipase A2 activity of naive and sensitized guinea-pig alveolar macrophages.

1. We studied the effect exerted by hr-interleukin-1alpha (IL-1alpha) on responsiveness of alveolar macrophages (AM) from naive and sensitized guinea-pigs, through O2.- production (by ferricytochrome C reduction), platelet-activating factor (PAF) release (by platelet aggregation), prostaglandin E2 (PGE2) release (by a radioimmunoassay), and cytosolic phospholipase A2 (cPLA2) activity (by hydrolysis of radioactive substrate). 2. In naive guinea-pig AM, 0.06 nM hr-IL-1alpha pretreatment decreased by 65% O2.- release stimulated with 10 nM fMLP. In contrast, O2.- production was not affected in sensitized guinea-pig AM. 3. O2.- release elicited by fMLP stimulation in both cell groups was affected by PLA2 inhibitors (10 microM bromophenacyl bromide, BPB or 10 microM methylprednisolone, MP). In contrast, 10 microM arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, was ineffective. 4. In naive AM, PAF release was elicited by hr-IL-1alpha pretreatment and by separate fMLP-stimulation, but when the stimulus was added to hr-IL-1alpha-pretreated cells inhibition of PAF release was observed. In sensitized AM, PAF release was lower than that found in naive guinea-pig AM in both hr-IL-1alpha-pretreated and fMLP-stimulated cells. 5. PGE2 release was unaffected by hr-IL-1alpha pretreatment and it was decreased by fMLP in both naive and sensitized AMs. The latter released less PGE2 than naive cells in basal conditions and after fMLP treatment. 6. Sensitized AM showed a greater cPLA2 activity in all experimental conditions in comparison to naive cells. cPLA2 activity assayed in the cytosolic fraction was found to be enhanced by hr-IL-1alpha pretreatment and by fMLP stimulation in naive but not in sensitized AM. However, when the stimulus was added to hr-IL-1alpha-pretreated cells we observed a decrease in cPLA2 activity in the cytosol and an increase in the membranes, thus suggesting a translocation of enzymatic activity. 7. In conclusion, hr-IL-1alpha can modulate the responsiveness of AM from naive and sensitized guinea-pigs, as suggested by changes found in the release of PAF and O2.- and in cPLA2 activity; therefore, sensitization itself may affect cellular responsiveness.

Hemoglobin affects lipid peroxidation and prostaglandin E2 formation in rat corticocerebral tissues in vitro.

Variations of lipid peroxidation and arachidonic acid (AA) metabolism products were found when experimental subarachnoid hemorrhage or ischemia and reperfusion were performed in an animal brain model. In a previous study, we showed that hemoglobin (Hb) produces prostaglandins when incubated in AA. To elucidate how Hb affects lipid peroxidation and AA metabolism in the CNS, we measured lipid hydroperoxides (LOOH), PGE2 and thiobarbituric acid reactant substances (TBARS) in corticocerebral homogenates and slices of rats (normal rats) after incubation with different concentrations (10(-9) to 10(-5) M) of Hb. In addition, brain cortices of indomethacin-treated (40 mg/Kg) rats (IN-treated rat) were incubated in the presence of 10(-5) M indomethacin (IN) to exclude the interference of prostaglandin enzyme synthetase. Hb was able to affect LOOH, PGE2, and TBARS production in both normal and IN-treated rat brain cortex homogenates and slices. In all cases, we found an increase in prostaglandin when 10(-8) M Hb was used, whereas no effect was noticed with 10(-9) M. On the other hand, with higher Hb concentrations (10(-6)-10(-5) M), the LOOH and PGE2 values did not reach statistical significance, and TBARS significantly increased. In all cases, when 10(-4) M scavenger or metal-chelating compounds were added to an incubation mixture with 10(-8) M Hb, PGE2 formation was inhibited, whereas no variation occurred when 10(-4) M IN was further added to IN-treated rat corticocerebral homogenate or slices. We hypothesize that in in vivo experimental neuropathologies, Hb must attain the 10(-8) M concentration in the reaction cellular microenvironment to stimulate PGE2 production, and that an evaluable part of this PGE2 production may be directly ascribable to the iron-heme oxy-redoxy activity of Hb.

Endoscopic stent placement for cancer of the lower esophagus and gastric cardia.

We reviewed our results of using stents for palliation of cancer of the lower third of the esophagus and gastric cardia. During a 14-year period, 76 patients with either lower third esophageal cancer (n = 43) or cancer of the gastric cardia (n = 33) received stents for palliation of malignant dysphagia. Successful endoscopic placement was initially achieved in all patients, with 71 patients available for follow-up. Of these, 40 (56%) were subsequently able to eat solid or semi-solid food, 25 (35%) could swallow only liquids, and 6 (8%) were unimproved. The combined early and late complication rate totalled 22%. Early complications included perforation (n = 3) and stent migration (n = 4); late complications consisted of dislodgment (n = 6), obstruction by tumor (n = 2), and severe esophagitis (n = 1). There were no procedure-related deaths, but survival at 1 year was estimated to be only 1.5%, with a median survival of 2.5 months after stent insertion. The endoscopic placement of prosthetic stents for cancer of the distal esophagus and gastric cardia entails a higher complication rate, less successful palliation, and shorter survival time compared to similar treatment for more proximal esophageal cancer.

Effect of 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bis(diethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl-, HCl (16 alpha)) on rat brain cortex lipid peroxidation induced "in vivo" by iron-carbohydrate.

Compounds derived from glucocorticoids, 21-aminosteroids, were reported to inhibit in vitro lipid peroxidation in CNS tissue. In order to evaluate the possible scavenging and/or iron chelating activities in vivo of the 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bisdiethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl- , HCl (16 alpha)), the drug was administered for seven days to rats. These rats had been induced by iron-saccharate complex injection a slow process of lipid peroxidation into their right brain hemicortex. The drug was injected also to intact rats (normal rats). Seven days after the operation the extent of iron-induced lipid peroxidation in both the hemicortices and the effect of the drug, were assessed by the evaluation of lipid-soluble fluorescence and of conjugated diene formation. The assessment was performed both in vehicle (control) and in U74500A-treated rats. In the iron-injected rat groups the drug induced a significant dose-related reduction of fluorescence values. Formation of conjugated dienes showed a significant decrease when U74500A (48 mg/kg every 48 hr) was administered to cortico-cerebrally iron-injected animals. The lipid peroxidation of cortices in normal rats was evaluated as thiobarbituric acid reactant substances in both the drug-treated and the control animals. In normal rats, U74500A (48 mg/kg every 48 hr) caused a significant decrease of TBARS values, as compared to those observed in the control group. The iron content in the iron-injected hemicortices, which was evaluated by the ferrozine method, was not modified by drug treatment. U74500A appears to have in vivo antioxidant properties and not to affect the iron content in the neural tissue. An interaction of this drug with the metal, however, cannot be excluded.

D-penicillamine affects lipid peroxidation and iron content in the rat brain cortex.

D-Penicillamine, a trifunctional amino acid known for its ability to form metal complexes and for being a radical scavenger, has been investigated "in vitro" and "in vivo" in the rat brain cortex. At 50 microM the drug facilitated lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of the D-penicillamine (25 and 50 mg/Kg i.p.) was evaluated "in vivo" after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated with D-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe2+/Fe3+ ratio was significantly different from that of normal rats. On the contrary ratios obtained form D-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest that D-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agent, can remove metal from action sites where lipid peroxidation may occur.

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

Lipid peroxidation induced "in vivo" by iron-carbohydrate complex in the rat brain cortex.

In view of the emerging role of metals and particularly iron in the pathogenesis of several ischemic or degenerative CNS diseases, via a lipid peroxidative process, a model of slow iron-induced peroxidative damage in the rat brain cortex has been carried out. Iron-carbohydrate complexes were injected in the right brain cortex, and biochemical assays were performed on ipsilateral and contralateral samples two hours or seven days after injection. Iron-saccharate caused a significant increase in the ipsilateral cortex in TBARS, conjugated dienes and fluorescent substances seven days after injection, whereas no biochemical alteration was observed two hours after treatment. In order to prevent or to limit lipid peroxidation, some drugs known for chelating and/or scavenging activity were administered to iron-injected rats. DL-alpha-tocopherol, methylprednisolone, D-penicillamine significantly decreased the value of fluorescent products formed by iron-saccharate, whereas desferrioxamine was not effective.

Capsaicin and anaphylactic reactions in the guinea-pig airways.

Further evidence is reported on the influence exerted by capsaicin on the anaphylactic reaction evoked in actively sensitized guinea-pigs. In Herxheimer microshock induced by ovalbumin aerosol, pretreatment of animals with 100 micrograms/kg i.p. capsaicin prolonged the preconvulsion time when the drug was administered 3 h before antigen challenge. In contrast, the same dose of capsaicin injected 30 min before aerosol caused a shortening of latency of the respiratory symptomatology. The influence of the drug is no longer evident after 24 h. In "in vitro" experiments desensitization to capsaicin of tracheal preparations caused a reduction of histamine and SRS-A released during antigen challenge, in comparison to controls. Moreover, anaphylactic histamine release was increased in preparations perfused with 10(-8) M substance P. In conclusion, our findings confirm that neuropeptides may be involved in the pathogenesis of asthma by affecting release of mediators.

Histamine release from rat mast cells induced by metabolic activation of polyunsaturated fatty acids into free radicals.

Polyunsaturated fatty acids (PUFA: arachidonic and linoleic acid) release histamine from isolated purified rat serosal mast cells only in the presence of oxidizing systems such as phenobarbital-induced rat liver microsomes, prostaglandin-H-synthetase (PHS) or soybean lipoxygenase. The release of mast cell histamine by activated PUFA has a long time-course and the electron microscopical features are consistent with an exocytotic secretion in the case of arachidonic acid and cell lysis in the case of linoleic acid. The phenomenon is associated with a significant increase in malonyldialdehyde (MDA) and conjugated diene generation, suggesting a relationship between histamine release and membrane lipid peroxidation. The secretion of histamine was inhibited by anti-free radical interventions such as D-mannitol, reduced glutathione and alpha-tocopherol. Some cyclooxygenase and lipoxygenase inhibitors, cimetidine and carnitine derivatives, are differentially active in the inhibition of mast cell histamine release by activated arachidonic acid. These results suggest that free radical derivatives of PUFA, generated by metabolic activation, trigger mast cell histamine release.

[Low-malignancy duodenal leiomyosarcoma]. / Il leiomiosarcoma duodenale a bassa malignità.

The authors report a case of duodenal leiomyosarcoma presenting a low degree of histological malignancy. The main anatomoclinical features of the neoplasm are summarized. The authors conclude indicating not aggressive surgical therapy as adequate in such cases. However, a prolonged post-operative follow-up is always necessary, in order to detect recurrences as early as possible.