Metabolic function of grafted liver in rats.

We studied the metabolic variations in grafted livers at different times after transplant by measuring the hepatic energy and redox states. Five groups of rats were studied: control ungrafted Wistar (RT1y) rats (group 1), ungrafted Wistar rats with ligature of the hepatic artery (group 2), isografted Wistar rats (group 3), allografted Wistar rats with livers from ACI (RT1a) donors (group 4, long-term surviving rat strain combination), and allografted Wistar rats with livers from BN (RT1n) rats (group 5, rejector rats). The metabolism of grafted livers was studied for 7 days in groups 2 and 3, for 2 months in group 4, and at the time of rejection in group 5. Adenine nucleotide levels (ATP, ADP, AMP) were significantly impaired at 24 hr and at 48 hr from grafting in isografted and in allografted livers, and the reestablishment of normal values began at the 7th day from grafting. Cytoplasmic NAD+/NADH ratios were lowered at 24 hr from grafting in isografted and in allografted livers. Mitochondrial NAD+/NADH ratios were lowered at 24 hr in isografted livers and at 24 hr and 48 hr from grafting in allografted livers. The metabolic studies performed for 2 months revealed a significant correlation between well-maintained metabolic functions and transplant survival. On the contrary, an important energy loss was evidenced in livers of group 5, at the time of rejection.

Protection of rat heart from damage due to ischemia-reperfusion during procurement and grafting by defibrotide.

We studied the efficacy of defibrotide, a prostacyclin-stimulating agent, in preventing ischemia reperfusion injury in Wistar rat heart by using three experimental models: (1) hearts from donors were perfused with the drug (32 mg/kg/hr) during 15, 30, 45, and 60 min of cold ischemia following 5, 10, and 15 min of warm ischemia; (2) hearts from donors treated with the drug were cold-stored for 12 or 24 hr; and (3) procured hearts perfused with the drug were isografted, after 30 or 60 min of warm ischemia, in recipient rats treated daily with defibrotide. Hearts perfused with saline and/or vehicle of the drug were used as controls. At the end of established ischemia times, and after 30 min, and 2, 4, 7 and 14 days from transplantation, hearts were rapidly cooled in liquid nitrogen. ATP, ADP, AMP, cAMP contents, and NAD+/NADH ratios were evaluated in prepared tissue extracts. Cardiac ATP and ADP levels and NAD+/NADH ratios were significantly higher in defibrotide-treated organs than in controls. Isografted defibrotide-treated hearts were also significantly preserved, with respect to controls, from the loss of ATP levels until rejection occurred. Our results demonstrate the protective activity of the drug against the myocardial metabolic damage due to ischemia-reperfusion.

Effect of cyclosporine and aminophylline on streptozotocin-induced diabetes in rats.

Diabetes induced in rats by multiple low doses of streptozotocin is thought to mimic type 1 disease in man. We tested the effect of concomitant treatment with immunomodulator drugs in this diabetic experimental model. Administration of cyclosporine resulted in a rapid appearance of hyperglycemia, perhaps by a potentiation of the direct cytotoxic action of streptozotocin on beta cells. By contrast, aminophylline administration protected the animals from the diabetogenic action of streptozotocin. Concomitant treatment with aminophylline and cyclosporine failed to protect the rats from the hyperglycemia induced by streptozotocin.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P < 0.05), and after endothelial cell stimulation (TNF-alpha, 500 u ml-1) or after leukocyte stimulation (fMLP, 10(-7) M), it inhibited leukocyte adhesion by 26.5% +/- 3.4 and 32.4% +/- 1.8, respectively (P < 0.05). 4. In adhesion blockage experiments, the use of the monoclonal antibody anti-CD31 (5 micrograms ml-1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti-LFA-1 (5 micrograms ml-1) significantly interfered with the effect of defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action.

Protection by L-2-oxothiazolidine-4-carboxylic acid of hydrogen peroxide-induced CD3zeta and CD16zeta chain down-regulation in human peripheral blood lymphocytes and lymphokine-activated killer cells.

We investigated whether L-2-oxothiazolidine-4-carboxylic acid (OTC) [in the form of Procysteine, kindly donated by Transcend Therapeutics] could protect peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells from CD3zeta and CD16zeta chain down-regulation induced by H2O2 produced by lipopolysaccharide (LPS)-activated autologous monocytes. OTC is known to enhance glutathione production in cells in which glutathione was depleted by reactive oxygen species. Our data showed that OTC induced a significant increase in CD3zeta and CD16zeta chain expression in peripheral blood lymphocytes and LAK cells, respectively, pretreated for 12 hr at 37 degrees. Moreover, OTC significantly protected peripheral blood lymphocytes and LAK against decreased zeta chain expression induced by lipopolysaccharide-activated monocytes or the addition of H2O2 to the culture medium. Our experiments thus suggested that alterations in signal-transducing molecules, such as decreased CD3zeta and CD16zeta expression observed in cytotoxic T lymphocytes and LAK cells in response to oxidative stress, could be prevented by the use of OTC.

Effect of somatostatin on beta-endorphin release in rat experimental chronic inflammation.

The aim of the present study was to investigate the effect of somatostatin administration in arthritic rats. Inflammation was induced by daily interplantar injection of 100 microl of Freund's complete adjuvant into the left hind paw of the rat. Arthritis developed 20 days following the first injection and was stable in the inoculate paw. Arthritic rats were treated interplantarly with somatostatin (5 or 10 microg) or with indomethacin (100 microg) daily for 14 days. Inflammatory response was studied at 12 h, 7 and 14 days following drug administration. The effect of somatostatin was determined by local (into popliteal lymph nodes) and systemic production of beta-endorphin. Our results showed that somatostatin treatment significantly increased beta-endorphin levels in the blood and lymphocytes from popliteal lymph nodes. Greater efficiency was seen when 5 microg instead of 10 microg of somatostatin was used. A significant decrease of absolute leukocytosis was observed at the 14th day following somatostatin administration. Moreover, a significant reduction of plasmatic beta-globulins at 12 h and the 7th day and of plasmatic alpha2-globulins at the 14th day was observed after the beginning of somatostatin treatment.

Antiischaemic effect of defibrotide treatment in rat kidney.

The authors previously demonstrated the protective activity of defibrotide (a profibrinolytic and antithrombotic drug) on endothelial cells. In the present work they examine the efficacy of defibrotide in protecting rat kidney from ischaemic/reperfusion injury by studying the modifications of intrarenal adenine nucleotide levels. Right renal ischaemia of 60 min and reperfusion of 30 min were induced in adult male Wistar rats. Defibrotide was administered as a bolus through a catheter inserted into the left femoral vein 5 min before the beginning of ischaemia at the dose of 32 mg/kg and continuously infused during ischaemia/reperfusion through the same vein at the final dose of 32 mg/kg in 5 ml of saline at the rate of 3 ml/h. Rats treated with vehicle of the drug were used as controls. At the end of postischaemic reperfusion, the ischaemic and left kidneys were rapidly removed and frozen in liquid nitrogen. Tissue extracts were prepared, and their ATP, ADP, AMP, cAMP, NAD+, and NADH contents were determined by using luminescence methods. In controls, ATP intrarenal levels were significantly higher in the left kidney than in the ischaemic organ of the same rat (3405 +/- 320 vs. 378 +/- 36 nmol/g fresh tissue and mean +/- s.e.m. of 10 experiments). Defibrotide treatment significantly protected ischaemic kidneys from the drop in ATP intrarenal content (1465 +/- 147 vs. 3124 +/- 303 nmol/g fresh tissue measured in the left kidney).

Effect of acute and chronic inflammation on plasma growth hormone levels in rats.

We investigated the effect of acute and chronic stress on growth hormone (GH) plasma levels in rats. Acute stress was provoked by intravenous administrations of IL-1 beta and TNF-alpha. Determinations were made at 10, 30, 60, 120 and 180 min following i.v. injection of these cytokines into the caudal vein. We also investigated the chronic stress induced by hind paw injections of Freund's adjuvant. Arthritis was developed by 21 days following such injection. GH levels were studied at 7, 14 and 21 days after induction of arthritis on several blood samples which were withdrawn from tail veins, and long-term hormonal profiles (3 hours' sampling) were determined at 12.00 am, 1.30 pm and 3.00 pm. Local administration of dexamethasone and the monoclonal antibody anti-ICAM-1 were also used in arthritic rats. Following acute stress, a significant reduction of plasma GH levels has been evidenced, possibly related to the stimulation of corticotropin-releasing hormone. Following chronic stress, we demonstrated a significant increase of GH levels, which were significantly reduced by dexamethasone treatment and to a lesser extent by anti-ICAM-1 administration.

Possible role of defibrotide in endothelial cell protection.

Defibrotide is a single-stranded DNA fraction obtained from mammalian lung and is able to increase prostacyclin production by endothelial cells. It has profibrinolytic and antithrombotic properties, and we have successfully used it as an anti-ischaemic drug in many in-vivo experimental models. In fact, we showed that defibrotide treatment significantly protected rat heart, kidney and liver from ischaemia and postischaemic reperfusion injury. In rats treated with defibrotide, the functionality and metabolic activity of ischaemic organs were significantly protected from impairment as compared to controls treated only with the vehicle of the drug. In the present work we evaluated all our results, together with those of others, in order to hypothesize the mechanism of action of the drug. We postulated that the prominent function of defibrotide is to inhibit platelet and leukocyte adhesion to endothelial cells: this may depend on reduced cell activation possibly following drug interaction with adenosine receptors. Defibrotide could also favour endothelial-cell function through binding with haemoglobin: such binding permits oxygen release and preservation of endothelium-derived relaxing factor. Moreover, prostacyclin production by endothelial cells is responsible for many drug activities and also limits superoxide radical generation.

Defibrotide, a stimulator of prostacyclin (PGI2) production, prevents the effects of ischaemic damage.

Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, has been demonstrated to exert profibrinolytic and antithrombotic activity through stimulation of vascular prostacyclin (PGI2) production. We studied the effect of defibrotide administration in protecting liver and heart from ischaemic and postischaemic reperfusion damage. Defibrotide was administered as an i.v. bolus (30 mg/kg) at the beginning of liver ischaemia and at the same dose continuously during 60 min of postischaemic reperfusion. ATP levels were significantly improved in livers of defibrotide-treated rats as compared to those obtained in livers of rats treated with vehicle of the drug. Intrahepatic cytoplasmic and mitochondrial NAD+/NADH ratios were higher in defibrotide-treated than in vehicle-treated animals. The hearts, isolated from rats according to the transplantation procedure, were subjected to different times of warm + cold ischaemia. During ischaemia, the hearts were perfused continuously with 60 mg/kg of defibrotide or vehicle of the drug. The loss of creatine phosphokinase and lactate dehydrogenase activities due to an increased ischaemia time was limited in defibrotide-perfused hearts. Intracardiac ATP and ADP levels were significantly higher in defibrotide-treated organs than in controls. Our results demonstrate the efficacy of defibrotide in protecting liver and heart from ischaemia.

The therapeutic potential of Aloe Vera in tumor-bearing rats.

Aloe Vera has been claimed to contain several important therapeutic properties, including anticancer effects. The effect of Aloe Vera administration was studied on a pleural tumor in rat. Growth of Yoshida AH-130 ascite hepatoma cells injected (2 x 10(5) in 0.1 ml) into pleura of male inbred Fisher rats was evaluated at different times (7th and 14th days). Data show that the use of Aloe Vera proved a therapeutic method, and that the present experimental model could be useful in the study of other therapeutics treatments in vivo.

Prostacyclin release from endothelial cells, induced by defibrotide treatment, favours the function of grafted rat hearts and kidneys.

Defibrotide, a single-stranded DNA fraction obtained from mammalian lungs and able to increase prostacyclin production by endothelial cells, has been shown to be efficient in protecting rat organs (heart, kidney and liver) from ischaemic damage. We studied the efficacy of the drug in preserving the function of rat heart and kidney submitted to isotransplantation. Defibrotide was administered to donor Wistar rats at the dose of 32 mg/kg in 1.5 ml of saline. Heart and kidney were isolated and cold-preserved in buffered phosphate medium and continuously infused with defibrotide (32 mg/h) through the innominate or renal artery. Recipient Wistar rats were treated with defibrotide before and after transplantation at the dose of 32 mg/kg/day. Controls were treated with the vehicle of the drug. The function of isografted organs was evaluated at 12 and 24 h and at 2, 4 and 7 days from grafting. Heart function was evaluated by studying creatinine phosphokinase (CPK) and lactic dehydrogenase (LDH) activities of myocardial tissue. Renal function was evaluated by studying serum creatinine and urea levels of kidney-grafted rats. CPK and LDH activities were found to be significantly higher in defibrotide-treated rats than in controls. Creatinine and urea levels remained significantly lower in defibrotide-treated rats than in the controls. The results of the present work indicate that defibrotide treatment is useful to maintain the functionality of grafted hearts and kidneys.

Are increased ATP blood levels responsible for PGI2 release from endothelium?

Defibrotide, a polydeoxyribonucleotide with profibrinolytic and antithrombotic properties obtained from mammalian lungs was shown capable of favoring the release of prostacyclin from endothelial cells. We previously evidenced that the use of defibrotide i.p. (32 mg/kg for 30 min) or orally (50 mg/kg for 1 h) induced a significant increase in 2,3-diphosphoglycerate (2,3-DPG) content of blood cells in rats. We tested the in-vivo capacity of defibrotide to modify over 90 min the blood content of ATP, cAMP and 2,3-DPG. Male Wistar rats were anaesthetized with pentobarbital (20 mg/kg) i.p.; the left carotid artery was cannulated with a polyethylene tube, from which 0.4 ml of blood was drawn at 0, 5, 10, 15, 20, 30, 40, 50, 60 and 90 min. At 0 min, defibrotide was administered i.p. or i.v. (through the femoral vein) at the dose of 1 ml containing 80 mg of drug or orally at the dose of 1 ml containing 160 mg of drug. The control rats employed received either the vehicle of the drug or physiologic saline. Our results demonstrated a significant drug-related increase with time (maximal levels were revealed about 20 min after i.v., i.p., or oral defibrotide treatment) of blood ATP content and a significant decrease in cAMP content as compared with the controls. Our data confirm the relation between increased drug-induced prostacyclin production and that of ATP of in blood.