Physiopathological effects of the NO donor 3-morpholinosydnonimine on rat cortical synaptosomes.

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO(-) generated and H(2)O(2) formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O(2) min(-1) mg(-1) protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O(2) min(-1) mg(-1). This decrease was not modified by catalase or Trolox, demonstrating that ONOO(-) was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line.

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Pulmonary catabolism of interferon-gamma evaluated by lung perfusion of both normal and smoke-exposed rats.

The role of the lungs in the catabolism of rat recombinant interferon-gamma, either in normal rats or in rats subjected to an acute cigarette smoking episode, was evaluated using an isolated and perfused lung preparation. After administration of interferon-gamma into the lung perfusion medium, there was no clearance of the cytokine in either control or smoke-exposed rat lungs, and only 0.1 +/- 0.2% of the total dose was recovered in the bronchoalveolar lavage fluid. When the same amount of interferon-gamma was instilled into the bronchial alveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 71.2 +/- 4.3 and 62 +/- 5.7% of the administered dose, as measured by ELISA test, had been transferred from the bronchial lumen to the perfusion medium of either control or smoke-exposed rat lungs, respectively, the latter values being significantly lower (p < or = 0.05) than those obtained in control lungs. Moreover, total recoveries of interferon-gamma evaluated in smoke-exposed rat lungs (78.4 +/- 8.6%) were also significantly lower than those observed in control rat lungs (91.4 +/- 11.8%). Biologic activity evaluations on the same samples gave values significantly lower than those obtained using ELISA, indicating a partial loss of biologic activity during transalveolar transit. In conclusion, it appears that the transfer of interferon-gamma is almost exclusively unidirectional from the alveolar space to the plasmatic pool, with partial degradation during transalveolar passage.

Interferon production in BCG-sensitized rabbits challenged with PPD.

Intravenous administration of PPD into BCG-primed rabbits results in the release of IFN into the circulation. Peak interferon titres occur in plasma 1 h after PPD injection, rapidly decline during the following 3 h and depend on the dose of tuberculin administered. Bilateral nephrectomy of BCG-primed rabbits challenged with PPD affects remarkably the disappearance of IFN from the circulation. Characterization of the BCG-PPD-induced IFN shows that it is heat-labile, is almost completely inactivated after dialysis at pH 2 and displays a remarkable cross-species activity, particularly on human cell lines.

Interferon induction in rabbits after intraduodenal administration of a phosphorylated glucomannan-protein fraction of the cell wall of Candida albicans.

The aim of this work was to demonstrate whether a glucomannan protein fraction (GMP) of Candida albicans cell wall could induce interferon after intraduodenal administration in normal rabbits and rabbits immunized against C. albicans. For this purpose we collected simultaneously plasma and abdominal lymph for 10 h after the administration of the inducer. We observed a peak of antiviral activity in the lymph 4 h after intraduodenal administration of 20 mg GMP dissolved in saline to 6 normal rabbits. Immunized rabbits (anti-GMP titres greater than 1024) responded earlier (peak after 2 h) and more intensely; analysis of the values of the areas under the curve indicated that the IFN response in the lymph of immunized rabbits was significantly higher (P less than 0.0025) than in normal rabbits. Antiviral activity was absent in plasma in all cases. Preliminary characterization of the IFN activity has shown it to be trypsin-sensitive, acid and heat stable, and species-specific.

Renal metabolism of homologous serum interferon.

The metabolic behaviour of homologous native and desialylated serum and urinary interferons has been investigated by using an isolated and perfused rabbit kidney, the performance of which is comparable to that in vivo. Serum native and urinary interferons disappear from the plasma perfusate with a fractional turnover rate of 1.8 and 2% and half-lives of 3 and 35 min, respectively. Serum desialylated interferon disappears much more rapidly in keeping with the finding that the glomerular sieve poses less steric hindrance and electrophysical repulsion to the passage of less anionic proteins. These results confirm and extend our previous findings, which indicated that the kidneys have a predominant catabolic role and can explain to some extent the rapid disappearance of interferon from plasma.

Pulmonary catabolism of interferons: alveolar absorption of 125I-labeled human interferon alpha is accompanied by partial loss of biological activity.

The catabolism of interferon was examined in isolated rabbit lungs which were ventilated and perfused with homologous blood. Natural human interferon-alpha (HuIFN-alpha) from lymphoblastoid Namalwa cells or recombinant DNA-derived HuIFN-alpha 2 were labeled with 125I, mixed with an excess of the respective cold interferons and added to the perfusion blood. Protein-bound and acid-soluble radioactivity, as well as antiviral activity, were measured at regular time intervals. During the first 3 h of perfusion, only very small fractions of the interferons disappeared from the perfusate, irrespective of whether lungs were inserted in the perfusion system. This indicated that catabolism of interferons in the pulmonary circulation was negligible. On the other hand, when the interferons were instilled into the bronchial-alveolar tree, absorption of antiviral activity differed from that of acid-precipitable protein-associated radioactivity. While most of the radioactivity was transferred into the perfusate, only 2% of antiviral activity of natural HuIFN-alpha and 30% of that of HuIFN-alpha 2 were recovered in the perfusate. In both cases acid-soluble radioactivity in the system reached about 10%. Since radioiodide, instilled in the bronchial-alveolar tree, was transported rapidly into the perfusate, this type of analysis did not help in locating the site(s) of degradation. Alveolar macrophages did not catabolize or inactivate interferons in vitro.

LDL and HDL affect nitric oxide metabolism in human astrocytoma cells.

Astrocytes provide structural, trophic and metabolic support to neurons and modulate synaptic activity. Under physiological conditions, neuronal-derived nitric oxide (NO) plays an important role in the modulation of a variety of central nervous system (CNS) functions. NO, although short lived, can travel sufficient distances to be able to act as an intercellular messenger in the brain. Its targets include adjacent neurons and astrocytes. The aim of the present study was performed in order to investigate the effects produced by incubation of lipoproteins, at different times, with human astrocytoma cells and thus measuring NO and its metabolite production. NO and peroxynitrite production, iNOS and nNOS expression by Western immunoblot were evaluated. The LDL and HDL-treated cells showed an increased production of NO, more evident after 12 h, compared to basal levels; concerning peroxynitrite production, LDL and HDL-treated cells showed a higher fluorescence, more evident at 3 h. nNOS and iNOS protein levels were significantly higher in the cells incubated with control LDL and HDL. The present work supports the hypothesis that lipoproteins can induce the formation of reactive astrocytes, inducing iNOS as reported by other authors, giving experimental support to a role played by LDL and HDL inducing a reactive response.

Production of tumor necrosis factor alpha by rat alveolar macrophages collected after acute cigarette smoking.

In this study we evaluated the effect of cigarette smoke on the activation of alveolar macrophages of the rat lungs exposed to an episode of acute passive cigarette smoking. Our experiments were carried out in rats that, after undergoing smoking (3 cigarettes within 1 h) showed a COHb increase of about 16%. The evaluation of the kinetics of alveolar and peritoneal macrophages, indicated that the number of alveolar macrophages in the bronchoalveolar lavage fluids significantly increased 8 h after the smoking session, whereas the number of peritoneal macrophages remained practically constant. Alveolar macrophages collected 0.8 and 24 h after smoking and incubated for 24 h at 37 degrees C in an atmosphere of 5% CO2 in air spontaneously released 5 +/- 1, 48 +/- 14 and 15 +/- 9 units of TNF-alpha per 10(6) cells, respectively. Moreover, neither alveolar macrophages collected from smokers, nor those collected from controls, released IFN, and both cytokines were also absent either in bronchoalveolar lavage and peritoneal lavage fluids or in plasma. Alveolar macrophages collected from controls rats, when challenged with lipopolysaccharide (LPS), released more TNF than those collected from smoke exposed rats. Thus, it seemed that macrophages of experimental animals were activated but at the same time were somewhat depressed and responded less well to LPS.

The lymphatic route. VII. Distribution of recombinant human interleukin-2 in rabbit plasma and lymph.

Human recombinant (R) interleukin-2 (IL-2) has been administered through intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) routes and its distribution in lymph and plasma has been evaluated in rabbits. It has been shown that after i.m. administration of RIL-2 in saline, the lymphokine is preferentially absorbed via lymphatics. A similar result has been obtained after s.c. administration when RIL-2 was injected with a high concentration (12.5%) of human albumin, which acts as a retarder and a promoter of lymphatic absorption. These routes may be valid alternatives to i.v. administration.

Enhanced induction of plasma interferon after subcutaneous administration in rabbits of poly ICLC with albumin.

Intravenous administration of poly ICLC in rabbits yields very high plasma interferon (IFN) levels; dosages from 250 to 1000 micrograms/kg body weight are very toxic. Subcutaneous administration of poly ICLC together with a high concentration of human serum albumin potentiates the inducer activity in comparison to poly ICLC in saline. Finally, a dosage of 50 micrograms of poly ICLC in 12% serum albumin is more effective as an IFN inducer than other dosages.

Pulmonary catabolism of interleukin 6 evaluated by lung perfusion of normal and smoker rats.

Cytokines such as interleukin 6 are involved in the pulmonary inflammation arising as a result of smoking. By use of isolated and perfused lung preparations we have evaluated the role of the lungs in the catabolism of human recombinant interleukin 6 both in normal rats and in rats subjected to an acute cigarette smoking episode. When interleukin 6 was incorporated into the lung perfusion medium, neither control nor smoke-exposed rat lungs cleared the cytokine and only 0.1 +/- 0.2% of the total dose was recovered in the bronchoalveolar lavage fluid. When, on the other hand, the same amount of interleukin 6 was instilled into the bronchoalveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 70.1 +/- 9.8% and 40.9 +/- 22.5% of the administered dose, as measured by immunoenzymatic test, had been transferred from the bronchial lumen to the perfusion medium of either control or smoker rat lungs, respectively, indicating significantly (P < or = 0.05) different behaviour of the cytokine in the two experimental groups. Total recoveries of the administered interleukin 6 evaluated in smoke-exposed rat lungs were 55.3 +/- 23.2%, significantly lower than those for control rat lungs (83.9 +/- 11%). Determination of biological activity gave values always lower than those measured by immunoenzymatic test, indicating loss of biological activity during the transalveolar transit. It appears that the transfer of interleukin 6, especially in smokers, is almost exclusively unidirectional, from the alveolar space to the plasmatic pool with degradation during the transalveolar passage.

The lymphatic route. IX. Distribution of recombinant interferon-alpha 2 administered subcutaneously with oedematogenic drugs.

We have evaluated whether the addition of either bradykinin or histamine favours the lymphatic absorption of human recombinant interferon-alpha 2 (IFN-alpha 2) administered by the subcutaneous route. Subcutaneous administration of IFN-alpha 2 with bradykinin enhances IFN absorption via both capillaries and lymphatics, so that either the plasma or lymph areas under the concentration curves (AUC) increase significantly up to 1751 +/- 483 and 1319 +/- 608 IU/ml/min respectively as compared to the respective AUC values (613 +/- 208 and 483 +/- 213 IU/ml/min) obtained after IFN injection in normal saline. Since the lymph AUC/plasma AUC ratios remain unaltered, there is no preferential lymphatic absorption of IFN-alpha 2 after bradykinin administration. Dual-label experiments, 125I-IFN-alpha 2 in saline and 131I-IFN-alpha in saline containing 200 micrograms histamine were injected subcutaneously into the left and into the right shank of the same animal, gave similar results. The kinetics of 125I and 131I acid-soluble radioactivity confirm that histamine favours both plasmatic and lymphatic absorption.

Bulk-preparation of hepatic binding protein.

A method for the extraction in the cold of hepatic binding protein using a novel combination of detergents, namely 1% Na urso deoxycholate and 5% Tween 80 in the extraction buffer, which allows a significantly higher recovery of lectin than 1% Triton X-100 is described. It is noteworthy that this lectin preparation can be maintained in solution for over a month and can be used in vitro and in vivo.

Fatty acid composition of erythrocyte and vesicle total lipids during storage of human erythrocytes in protein-free media and in citrate-phosphate-dextrose.

We have studied the fatty acid composition of total lipids of erythrocytes and vesicles either during storage of human erythrocytes in their own plasma under blood bank conditions or during incubation at 37 degrees C in protein-free media. Vesicles appear as a heterogeneous population with a diameter of about 130 nm and a varying content of hemoglobin. The fatty acid pattern of vesicle total lipids changes in respect to control erythrocytes, while in erythrocytes it remains fairly constant during the total ageing period. Our results indicate a marked rearrangement of the membrane components during blood storage.

Short circulatory residence of the hepatic binding protein.

We evaluated the distribution and fate of homologous radioactive Hepatic-binding-protein (125I-HBP) in the rabbit after intravenous (iv) injection and the possibility that this protein may induce interferon production. We demonstrated that only 9% of the injected 125I-HBP remained in the circulation 30 min after injection. The 125I-HBP-Asialoorosomucoid complex displayed a longer half-life than the HBP alone, while 125I-Asialoorosomucoid had a very short half-life and only 1% of the dose was found in the circulation after iv administration. Ten min after iv injection of 125I-HBP the major amount of radioactivity was present in the liver and less in the kidneys and lung. HBP, after iv administration, does not stimulate interferon production and this fact is probably due to its rapid catabolism.

Spin label studies of erythrocytes during storage of blood.

The kinetic behavior of the spin label MAL-6 in the interaction with differently aged human erythrocyte membranes was evaluated by monitoring the rate of disappearance of the room temperature ESR signal due to the MAL-6 spin label added to blood after storage at 4 degrees C or after incubation of red cells at 37 degrees C in a protein-free medium. After 35 days of blood storage or 60 h of erythrocytes incubation at 37 degrees C the decrease of the intensity of the MAL-6 ESR spectra in respect to control samples is markedly enhanced and the correspondent kinetic constants significantly increase. Signal decay of MAL-6 is a further proof that during storage of blood under blood bank conditions or during an artificial ageing of erythrocytes at 37 degrees C, profound modifications occur in the human erythrocyte membrane.