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We discuss Gaussian fluctuations in a spin-glass model with one replica symmetry breaking (RSB) and we show how non-perturbative fluctuations of the break-point parameter can be included in the longitudinal propagator within linear response theory.

Nine polymorphic microsatellite loci for the pied flycatcher (Ficedula hypoleuca),from a wild population in Spain are isolated and their variability escribed on 70 individuals. The number of alleles per locus ranged from 6 to 41 and observed heterozygosity ranged from 0.75 to 0.98. These markers are being used to study mating strategies in Ficedula hypoleuca iberiae.

Resveratrol, a phytoalexin found in grapes and wine, has been shown to have anti-inflammatory properties. Since endothelium is activated during inflammation by some cytokines released by macrophages and many other cells, we tested whether resveratrol could modulate endothelial cell activation. We studied the effect of resveratrol treatment in vitro on the expression of vascular cell adhesion molecule-1 by tumour necrosis factor alpha-stimulated human umbilical vein endothelial cells. In addition, we studied the effect of resveratrol treatment in vivo (in a murine experimental model) on the modulation of tumour necrosis factor alpha-induced vascular permeability. Resveratrol, used at the concentrations present in human plasma following moderate wine consumption, was demonstrated to be an inhibitor of the adhesion molecule expression by tumour necrosis factor alpha-stimulated endothelial cells. In addition, resveratrol significantly prevented the cytokine-induced vascular leakage. Our results (both in vitro and in vivo) may explain some aspects of the anti-inflammatory effects of resveratrol.

There is increasing evidence that genotype affects pain sensitivity and pain modulation. As an example in humans, 10% of a Caucasian population studied only poorly metabolized the liver isozyme P450IID6 (required for the O-demethylation of the widely used opiate drug codeine to morphine) [1]. So, for such people codeine is an inefficient analgesic. The enzymatic defect is related to a mutation in the CYP2D6 gene [2]. As an experimental example, a recent study in rats demonstrated that a form of stress-induced analgesia was naloxone-insensitive, but attenuated by dizocilpine in male C57BL/6J mice. The same type of analgesia in male DBA/2J mice was significantly attenuated by naloxone but was insensitive to dizocilpine antagonism, indicating the role exerted by the different rat strain (i.e., genetic factors) in determining the selective recruitment of alternative central mechanisms of pain inhibition [3].

Herein we show that exposure of human umbilical vein endothelial cells to tumor necrosis factor alpha (TNFalpha) led to platelet endothelial cell adhesion molecule-1 (PECAM1) surface redistribution, disruption of cytoskeleton connections, and increased PECAM1 phosphorylation, accompanied by increased permeability to macromolecules. The in vitro use of inhibitors of tyrosine or serine-threonine kinases could prevent both PECAM1 surface redistribution and the increase in permeability induced by the cytokine. In vivo administration of lavendustin A, a natural tyrosine kinase inhibitor, protected endothelial cells from TNFalpha-dependent vascular leakage in mouse liver. We propose that the involvement of PECAM1 in TNFalpha-mediated effects on vascular permeability may depend on a dynamically regulated cytoskeletal association, related to the degree of PECAM1 phosphorylation.

Defibrotide (a polydeoxyribonucleotide) and oligotide (an oligodeoxyribonucleotide) obtained from mammalian single-stranded DNA, have been demonstrated to have anti-ischemic activity in some experimental models of ischemia/reperfusion of kidney in rats. We hypothesized that their anti-ischemic activity could be related to an inhibition of leukocyte-endothelial cell adhesion and also the consequent generation of oxygen free radicals by leukocytes. We studied the in vitro adhesion of neutrophils to human umbilical vein endothelial cells under basal conditions and following neutrophil or endothelial cell activation (using 10(-7) fMLP and 500 U/ml TNF-alpha, respectively). Defibrotide and oligotide significantly inhibited neutrophil adhesion to endothelial cells (after only 1 min of drug treatment). When the anti-LFA-1 70H12 F(ab)2 monoclonal antibody was used, the drugs exerted only slight additional inhibition of the adhesion of fMLP-activated neutrophils to endothelium. These results, confirmed in NIH/3T3-ICAM-1-transfected cells, demonstrate that defibrotide and oligotide interfere with leukocyte adhesion to endothelial cells by an LFA-1-dependent mechanism.

We previously demonstrated that anaesthetics affect leukocyte response by inhibiting lymphocyte capping and metabolism and the phagocytic activity of neutrophils in some experimental models. In the present study, we investigated the effects of the clinical use of three different types of anaesthesia on lymphocyte subset distribution and activity: inhalational anaesthesia (isoflurane), a neuroleptoanalgesia, or local anaesthesia was used in patients submitted to the same operation (hysterectomy). At time 0 (before administering the anaesthesia) and at 10 min and 48 h from the start of the operation, heparinized blood was obtained from each of the 30 patients studied; three lymphocyte subpopulations were isolated (CD4+, CD8+ and CD19+). Cell number was identified for each lymphocyte subset, and (as an indication of cellular function) the intracellular contents of ATP and cAMP were evaluated by luminescence methods. The relevant results for each of the 3 anaesthetic methods were as follows: i) treatment with isoflurane induced a significant reduction in the number and function of CD4+ cells at 10 min, which was reversed at 48 h; a functional but not reversible decrease of CD19+ cells was obtained, ii) treatment with neuroleptic drugs induced a significant progressive functional impairment of CD4+ and CD19+ cells, and iii) local anaesthesia caused a significant functional impairment of CD8+ cells at 48 h and a significant functional impairment of CD19+ cells at 10 min and 48 h.

By studying the structure of infrared divergences in a toy propagator in the replica approach to the Ising spin glass below $T_c$, we suggest a possible cancellation mechanism which could decrease the degree of singularity in the loop expansion., Comment: 13 pages, Latex , revised version

Local therapy with trichoriboside and trichosaccharide, which have been found to be beneficial for scalp hair maintenance in adult males affected by androgenic alopecia, was found to increase cAMP levels in human scalp hair follicles. The increase was significant in men affected by androgenic alopecia, whereas it was not significant in unaffected control men. Trichoriboside showed a greater activity than trichosaccharide, and such activity was accompanied by a significant concomitant reduction of ATP in the hair.

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).

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 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.

The effect of defibrotide treatment in protecting liver metabolism from ischemic damage was studied. The drug was administered to male Wistar rats as a bolus (30 mg/kg body weight) at the beginning of 60 min ischemia and then continuously during 60 min of postischemic reperfusion at a dose of 30 mg/kg body weight. This dose was previously identified as useful to protect against myocardial ischemia induced in the cat. ATP and ADP intrahepatic levels were significantly higher in drug-treated rats than in untreated animals. The liver cytoplasmic NAD+/NADH ratio in defibrotide-treated rats was no different from that observed in sham-operated rats. The mitochondrial NAD+/NADH ratio in the liver was also improved by defibrotide treatment. Our data suggest that defibrotide may exert protective activity on hepatocytes useful for inducing a rapid restoration of their metabolism. Such a restoration is possibly related to improvement of microcirculation through an increase in prostaglandin I2 production or oxygen delivery due to drug administration.

Previously labeled smooth-membranes, but not rough membranes, of the endoplasmic reticulum obtained from ischemic rat liver cells lose their radioactivity during long-duration ischemia that will ultimately cause cell death and necrosis. In the period following the re-establishment of the blood supply, irrespective of the duration of previous ischemia, smooth membranes recover their normal levels of radioactivity; at the same time, the specific radioactivity of the rough membranes shows a consistent decline. In post-ischemic livers, de-novo synthesis of membrane protein shows a lag phase of about 2 hours, and is then normally resumed in reversibly injured liver cells; it does not recover appreciably in cells eventually committed to necrosis. In agreement with this observation labeling of the nascent peptide chains on post-ischemic liver ribosomes recovers, after a lag period, only when cells have not been injured to death. Amino acid pools, expanded in ischemic livers, return to normal in both reversibly and irreversibly injured cells and in the former ones normal amino acid concentrations are attained at a time when synthesis of nascent chains is still impaired. These results seem to suggest that flow of the label along the membranes and size of the amino acid pools are not related to the occurrence of cell necrosis; on the contrary, synthesis of nascent peptide chains and of intrinsic membrane protein are better correlated with the final destiny of the cell and their resumption may be tentatively interpreted as the beginning and the necessary prerequisite of cell repair after injury.

There is still no reliable early indicator of pancreatic rejection. This constitutes the main reason why many groups favor simultaneous kidney and pancreas transplantation on the assumption that pancreatic rejection may be treated in time by monitoring the renal allograft course. To provide an experimental model to investigate this problem, we have developed a technique of simultaneous kidney and pancreas transplantation in the rat. In isografts functional evaluation of the grafted organs was performed by testing blood glucose, serum insulin, blood urea and intravenous GTT. Our results demonstrated that functional and histological evaluations of kidney and pancreas simultaneously grafted in rats were well maintained up to 9 months and were similar to those revealed in kidney or pancreas isografted alone. The experimental model of simultaneous kidney and pancreas transplantation here described appears useful to study pancreatic rejection in allograft combinations as well as for testing immunosuppressive treatments.

The in vivo effect of theophylline on lymphocyte nucleotide metabolism was investigated. Rat peripheral blood lymphocytes presented more elevated levels of adenosine 5'-triphosphate (ATP) and cyclic adenosine 3',5'-monophosphate (cAMP) than shown by spleen lymphocytes. A continuous (15 days) daily oral treatment of the rats with aminophylline increased intralymphocytic cAMp content in peripheral blood (by 14 times) and in spleen lymphocytes (by 4 times), whereas ATP content was not significantly changed. NAD+ and NADH levels and NAD+/NADH ratios appeared higher in peripheral blood than in spleen cells, and were not influenced by aminophylline treatment. These results may be explained by assuming the existence of a different distribution of lymphocyte subpopulations and of a different sensitivity of the same to aminophylline treatment. The studies of nucleotide variation in peripheral blood lymphocytes incubated at 37 degrees C in anoxic conditions (N2+CO2, 95:5) revealed an impairment of ATP production and a well-preserved redox state.

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.

Many reports indicate that anaesthesia affects several immunological functions that decrease the immune response, but the mechanisms involved are still unknown. We investigated the in vitro effect of halothane on human lymphocyte metabolism and plasma membrane function by evaluating the intracellular concentration of 3',5'-cyclic adenosine-monophosphate (cAMP), phosphodiesterase enzyme activity, NAD+/NADH intralymphocytic ratios and the degree of antibody and lectine-induced 'capping' of surface markers. Our results demonstrated an impaired lymphocyte capping of surface immunoglobulins and concanavalin A receptors 60 min after exposure to halothane at the concentration of 1% in oxygen. This phenomenon was reversible after 24 h and it was unrelated to the presence of adherent cells during the culture. Furthermore, halothane was able to induce a persistent increase in cAMP intracellular concentrations, which was reversible within 48 h. This effect was not dependent on adherent cells or on phosphodiesterase enzyme inhibition. Finally, no alteration in NAD+/NADH ratios after halothane exposure was observed.

A new surgical technique of simultaneous kidney and pancreas transplantation is described here. It was used to perform 28 isografts in inbred male Wistar ( RT1y ) rats: 3 animals died within 3 days from early complications and 4 had long-term complications (2 severe pancreatitis and 2 urinary tract complications); 1 animal was killed at 3 months, and its organs were examined histologically. At greater than 4 months after transplantation 2 animals died normoglycemic from undetermined causes. In 3 animals the isografted pancreas was removed at 6 months to assess the effectiveness of the transplant. Currently, 15 animals are alive and normoglycemic greater than 6 months after transplantation. Morphological aspects of simultaneously isografted organs are unchanged when compared with separately transplanted organs. In our opinion this technique provides a useful experimental model to study several technical and immunological problems still present in kidney and pancreas transplantation in diabetic patients.

Theophylline reversibly inhibits E rosette formation by a portion of human circulating T lymphocytes. We investigated the effect of theophylline on E rosette formation and intracellular content of cyclic nucleotides (cAMP, cGMP). When the amine is added to 15 human healthy donors' lymphocytes either before or after 24 hours of culture at 37 degrees C, in absence of mitogens, a portion of theophylline-sensitive T cells spontaneously becomes theophylline-resistant after 24 hours of culture. While the intracellular content of cAMP does not significantly vary, the ability of theophylline to induce an increase of cAMP appears to be impaired after 24 hours of culture. The possible correlation between theophylline resistance and impaired turnover of cAMP in the cultured lymphocytes is discussed.

Yoshida ascites hepatoma cells growing in vivo are characterized by a large number of monosomes and dimers which further increase when the cells are incubated anaerobically in vitro in the absence of glucose: under the latter condition most of the ribosomes are present as inactive particles. These monosomes can be incorporated into polyribosomes if the elongation step of protein synthesis is inhibited by cycloheximide. The degradation of polysomes in the absence of glucose under anaerobic conditions can be reversed promptly on addition of glucose to the medium and less promptly by oxygenation. These data suggest that the large monosome-dimer pool of hepatoma cells depends on a relative inefficiency of the initiation step of protein synthesis, which is magnified by the lack of energy caused by anaerobic incubation in the absence of glucose, and counteracted by interference with further steps of protein synthesis or conditions which favor glycolysis or, less efficiently, respiration of the tumor cells.

Acute intracerebroventricular injection of 25 micrograms capsaicin or 40 micrograms substance P in isotonic saline elicited approximately similar effects on the micturition reflex, but capsaicin had twice as much effect as substance P. This effect is specific, since acute intracerebroventricular injection of isotonic saline did not produce the micturition reflex. It can be hypothesized that capsaicin and substance P may act on the brain micturition centers directly or by mediation of neuropeptides such as tachykinins, but other hypotheses are also made.

Trans-resveratrol, a natural stilbene present in wine and grapes, has been studied mainly for its antiinflammatory and anticancer activities. In this study the activity of resveratrol on proliferative immunological parameters (differentiation, apoptosis, phagocytosis and intracellular killing) was studied using a U937 human promonocytic cell line in comparison with another polyphenol, quercetin. After incubation of the pathogen, Candida albicans, intracellular killing by macrophage-like cells was decreased by quercetin and resveratrol 10 microM but was enhanced by resveratrol 1 microM after 20 h of treatment. Phagocytosis rate, expressed as phagocytosis frequency, (i.e., percentage number of phagocytosing cells/total cells) at 20 h was highest with resveratrol 10 microM and was higher with quercetin 10 microM than with resveratrol 1 microM. The phagocytosis index exhibited the same trend. While both polyphenols demonstrated cytostatic activity on U937 growth, a prointraphagocytic effect for resveratrol 10 microM-treated cells at 10 min, resveratrol 1 microM-treated cells at 20 h and resveratrol 10 microM-treated cells at 48 h was observed. Morphological examination with optic microscopy demonstrated both apoptotic and differentiating cells, even after 10 min treatment. Resveratrol-induced apoptosis (following 4 h treatment) was confirmed by flow cytometry at concentrations as low as 1 microM and 100 nM in the assay for detection of membrane phosphatidylserine. Resveratrol- or quercetin-treated, but unstimulated cells, did not produce tumor necrosis factor-alpha protein. As phosphatidylserine externalization triggers specific recognition by monocytes and macrophages, removal of intact apoptotic cells is important a) in cell population selection and differentiation for antiblastic therapy, and b) in preventing the release of toxic inflammatory substances such as reactive oxygen substances and proteolytic enzymes by dying cells. This observation suggests that wine polyphenols, at the same concentrations as those found in plasma after moderate wine consumption, are important cofactors in antiinfective, antiinflammatory and anticancer nonspecific immune reactions.