Defective inflammatory response in interleukin 6-deficient mice.

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.

Peripheral benzodiazepine receptor ligands in rat liver mitochondria: effect on 27-hydroxylation of cholesterol.

The effect of peripheral benzodiazepine receptor ligands: PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamid e), Ro 5-4864 (4-chlorodiazepam), hemin, N-methyl protoporphyrin IX and protoporphyrin IX on liver mitochondrial 27-hydroxylation of cholesterol was studied by adding them together with [4-14C]cholesterol. N-Methyl protoporphyrin IX, PK11195 and protoporphyrin IX stimulated mitochondrial 27-hydroxylation of [4-14C] cholesterol in vitro, the first two being the most potent (2-3-fold increase). Ro 5-4864 and hemin were not active. 27-Hydroxylation of [4-14C]cholesterol was reduced to below control levels (respectively 40 and 56% decrease compared to control, P < 0.01) when PK11195, N-methyl protoporphyrin IX or protoporphyrin IX were allowed to equilibrate in vitro with mitochondria for 20 min at 37 degrees C. Hepatic protoporphyria was induced using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (100 mg/kg, i.p.) to study the effect of in vivo accumulation of large amounts of dicarboxylic porphyrins, i.e. endogenous peripheral benzodiazepine receptor ligands, on cholesterol 27-hydroxylation. DDC treatment caused an increase in total porphyrin content in liver homogenate (10-fold) and mitochondria (2-fold). Mitochondrial 27-hydroxylation of [4-14C]cholesterol was depressed after treatment (60% decrease, P < 0.01). We suggest that peripheral benzodiazepine receptor ligands act on liver mitochondrial 27-hydroxylation of cholesterol by a mechanism coupled to these receptors and that the time of exposure of peripheral benzodiazepine receptors to ligands is a major factor. The modulation of 27-hydroxycholesterol production may have a physiological role in liver and possibly in other tissues.

Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice.

Interleukin-2 (15 micrograms/mouse, i.p. twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased. The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde. In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked. Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor. This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450. Dexamethasone, already used in patients to reduce the toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression.

[Instrumental diagnosis of the post-phlebitic syndrome of the lower limbs]. / Contributo diagnostico-strumentale alla sindrome post-flebitica degli arti inferiori.

A series of patients with post-phlebitic syndrome (PPS) of the lower extremities is described with emphasis on the value of combined doppler echography and phleboscintiscans for diagnostic purposes. On this basis, PPS is classified into 5 stages. A personal treatment protocol validated by a two-year follow-up is also proposed.

[Post-stenotic aneurysm following incarceration of the popliteal artery. Description of a case]. / L'aneurisma post-stenotico da incarceramento dell'arteria poplitea. Descrizione di un caso.

The paper reports a case of post-stenotic aneurysm following an Inshua type II incarceration of the popliteal artery which was treated surgically, and associated with concomitant popliteal venous thrombosis, with complete remission of all clinical symptoms. The paper underlines the rarity of this pathology and the even rarer association with homolateral thrombosis of the popliteal vein. Anomalies which may occur following the incarceration of the popliteal artery are discussed, as are the causes for dilatory degeneration.

[The superior mesenteric artery syndrome. Anatomo-clinical study and therapeutic possibilities in vascular compression of the duodenum]. / La sindrome dell'arteria mesenterica superiore. Contributo allo studio anatomo-clinico e possibilità terapeutiche delle compressioni vascolari del duodeno.

Four cases of superiore mesenteric artery syndrome are presented. Reference is made to the difficulties involved in the clinical diagnosis of this form. It is pointed out that radiological examination is essential, since it dispel all doubts in nearly every case. Associations between the syndrome and peptic ulcer are relatively, and the joint surgical resolution of both forms is recommended.

[Kinetics of intravenously administered flecainide in patients with acute myocardial infarct]. / Cinetica della flecainide per via endovenosa in pazienti affetti da infarto miocardico acuto.

Flecainide (F) is a new antiarrhythmic agent recently introduced into clinical practice. The above study was aimed at evaluating its intravenous (iv) pharmacokinetics in patients (pts) with acute myocardial infarction (AMI) on 1st and 2nd day, complicated by complex ventricular premature beats (VPBs). 2 mg/kg F was given iv as bolus injection, followed by 300 mg/24 hrs iv infusion. Plasma F values were evaluated by HPLC. Plasma F levels increased progressively, in a non uniform but predictable manner: in pts with large AMI and cardiac failure, F plasma levels, although remaining within the therapeutic range, were greatly increased after the 2nd hour (P less than 0.05) in comparison with pts without cardiac insufficiency. Negative side effects, both cardiac and extracardiac, were not observed: F appeared a handy and effective agent in post-AMI arrhythmias, especially when plasma drug levels are controlled; plasma F level monitoring is anyway recommended in pts with cardiac failure, owing to the wide interindividual variations.

[Bleeding duodenal leiomyoma with secondary pseudodiverticulum]. / Leiomioma duodenale sanguinante con pseudo-diverticolo secondario.

A personal case of bleeding leiomyoma with secondary pseudo-diverticulum is presented. Attention is drawn to the comparative rarity of this form in the world literature and the marked difficulty raised by its diagnosis, especially if its frequent complications are absent. When such complications are present, it is felt that simple resection should give way to more radical management, such as d.gastric resection with gastrojejunal anastomosis.

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon beta in mice.

AIMS/METHODS: Interferon beta is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon beta on cytochrome P-450 in mice. RESULTS: Interferon beta (2 x 10(5) units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon beta reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In beta-naphthoflavone-treated mice, interferon beta lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). CONCLUSIONS: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon beta, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon beta.

Mechanisms of endotoxin-induced haem oxygenase mRNA accumulation in mouse liver: synergism by glutathione depletion and protection by N-acetylcysteine.

In in vitro systems haem oxygenase-1 (HO-1) mRNA increases after exposure to agents causing oxidative stress. We lowered cellular antioxidant defence systems in vivo by giving mice increasing doses (0.15 g/kg-1.6 g/kg) of DL-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis. Maximum glutathione depletion (80%) coincided with maximum hepatic HO-1 mRNA accumulation (about 20 times), whereas with 50% depletion, accumulation was only doubled. It has been suggested that reactive oxygen and nitrogen intermediates are involved in hepatic toxicity of endotoxin (lipopolysaccharide, LPS); LPS even at low doses [0.1 mg/kg, intraperitoneally (i.p.)] induces HO-1 mRNA about 25-fold after 1 h. Hepatic glutathione depletion (respectively 40% and 80%) after a low (0.3 g/kg) or a high (1.6 g/kg) BSO dose, resulted in potentiation of the HO-1 mRNA accumulation induced by LPS (0.1 mg/kg, i.p.). In the absence of BSO, N-acetylcysteine (NAC) (1 g/kg orally) reduced LPS-induced HO-1 mRNA accumulation to one fourth. Under the same experimental conditions S-adenosylmethionine (SAM) was not effective. NAC also reduced HO-1 mRNA accumulation when administered to mice in which glutathione was depleted and its synthesis blocked by BSO (1.6 g/kg). Thus reactive oxygen intermediates are likely mediators of LPS-induced HO-1 mRNA accumulation, and glutathione content appears to be one of the factors regulating this accumulation in the liver. Our findings are compatible with the theory that HO-1 induction might have a protective function in vivo when defence mechanisms against oxidants are challenged.

Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes.

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase nuclei can rapidly identify aneuploidies in uncultured amniotic fluid cells. Using DNA probe sets specific for chromosomes 13, 18, 21, X, and Y, we have identified 14 fetuses where the hybridization pattern was consistent with a triploid chromosome constitution. In each case, the identification of fetal abnormalities by ultrasound examination initiated a request for rapid determination of ploidy status via prenatal FISH analysis of uncultured amniocytes. FISH produced a three-signal pattern for the three autosomes in combination with signals indicating an XXX or XXY sex chromosome complement. This hybridization pattern was interpreted to be consistent with triploidy. Results were reported to the physician within 2 days of amniocentesis and subsequently confirmed by cytogenetics. These cases demonstrate the utility of FISH for rapid prenatal identification of triploidy, particularly when fetal abnormalities are seen with ultrasonographic examination.

Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils.

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.

Role of acute-phase proteins in interleukin-1-induced nonspecific resistance to bacterial infections in mice.

Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge of granulocytopenic and normal mice enhances nonspecific resistance. Since IL-1 induces secretion of acute-phase proteins, liver proteins which possess several detoxifying effects, we investigated the role of these proteins in the IL-1-induced protection. Inhibition of liver protein synthesis with D-galactosamine (GALN) completely inhibited the IL-1-induced synthesis of acute-phase proteins. GALN pretreatment abolished the protective effect of IL-1 on survival completely (neutropenic mice infected with Pseudomonas aeruginosa) or partially (nonneutropenic mice infected with Klebsiella pneumoniae). Pretreatment with IL-6, a cytokine induced by IL-1, did not reproduce the protection offered after IL-1 pretreatment, nor did it enhance or deteriorate the IL-1-enhanced resistance to infection. A protective effect of IL-1 via effects on glucose homeostasis during the acute-phase response was investigated by comparing plasma glucose levels in IL-1-treated mice and control mice before and during infection. Although glucose levels in IL-1-pretreated mice were somewhat higher in the later stages of infection, no significant differences from levels in control mice were present, and the glucose levels in control-treated animals never fell to hypoglycemic values. We conclude that the IL-1-induced nonspecific resistance is mediated neither by the induction of IL-6 nor by the effects of IL-1 on glucose homeostasis. Acute-phase proteins generated after IL-1 pretreatment, however, seem to play a critical role in the IL-1-induced protection to infection.

Evaluation of killing, superoxide anion and nitric oxide production by Leishmania infantum-infected dog monocytes.

Protozoa of the genus Leishmania infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. The parasitocidal mechanism of peripheral blood monocytes towards Leishmania in the dog has not been investigated in detail. Consequently, Leishmania infantum-infected monocyte cultures of healthy dogs were evaluated using the following parameters: (1) phagocytosis and killing capacities; (2) oxidative burst, in terms of superoxide anion (O2-) release, and (3) nitric oxide (NO) activity, in terms of nitrite (NO2-) production in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (NGMMLA). Parallel experiments were performed on monocytes stimulated with supernatants of concanavalin A-activated PBMC and on unstimulated monocytes. The amount of IFN-gamma in PBMC supernatants used for monocyte activation was determined by a biological assay on a canine Madin Darby cell line. Results demonstrated that phagocytosis, killing capacity and O2- production significantly increased in monocytes stimulated with supernatants, in comparison with unstimulated cells. A positive correlation was observed between the killing capacity, the O2- production and the amount of IFN-gamma in PBMC supernatants employed for monocyte activation. No significant differences were observed in NO production between unstimulated and stimulated cultures, or between the same cultures with and without NGMMLA. Finally, the killing percentage was similar in the presence or absence of NGMMLA, suggesting that in this experimental model peripheral blood dog monocytes lack NO-mediated killing.

[Endocavitary transcatheter electric fulguration as a therapeutic alternative in refractory ventricular tachycardia]. / La folgorazione elettrica endocavitaria transcatetere quale alternativa terapeutica della tachicardia ventricolare refrattaria.

Three patients affected by dilated cardiomyopathy complicated by refractory ventricular tachycardia, with a high risk of sudden cardiac death, underwent transcatheter electric fulguration. The technique was applied transeptally, using the terminals of two catheter electrodes as cathode and anode. These were placed at the right and left ventricular apex, at septal level where the "critical" arrhythmia point had been identified by endocardial mapping. All patients had previously experienced more than one episode of cardiac arrest and had successfully taken several antiarrhythmic drugs. All patients presented variable morphology of ventricular tachycardia (whether spontaneous or induced). In all of them clinical tachycardia was considered as having a left bundle branch block morphology with an earlier activation at low septal level. After treatment, antiarrhythmic therapy (amiodarone 200 mg/day) was continued for all patients, although at a lower dose than before fulguration. One patient has been free from sustained ventricular tachycardia for more than two years after fulguration. In the other patients we observed an early and late arrhythmic recurrence (respectively within 1 and 8 months following fulguration) in spite of antiarrhythmic therapy. The second patient presented no further recurrence after permanent pacemaker implantation. The third patient showed an arrhythmic recurrence, with a different morphology from the previous one, concomitantly with a septic process. This technique does not appear dangerous and may be used, in highly specialized centres, on carefully selected patients as a therapeutic approach after pharmacological therapy and before automatic defibrillator implantation or surgical antiarrhythmic intervention.

Depression of liver metabolism and induction of cytokine release by diphtheria and tetanus toxoids and pertussis vaccines: role of Bordetella pertussis cells in toxicity.

A 24-h pretreatment of mice with diphtheria and tetanus toxoids and whole-cell pertussis vaccines depressed liver cytochrome P-450 and therefore prolonged hexobarbital-induced sleeping time in mice. The depression of liver drug metabolism by a cellular vaccine containing a mutated pertussis toxin was less marked than that induced by the wild-type vaccines, indicating that the mutated vaccine might have lower toxicity in this regard. The wild-type vaccines decreased microsomal P-450 levels by 50%, while the mutated whole-cell vaccine had a less marked effect (a decrease of 30%), paralleling the results obtained in sleeping time experiments. Furthermore, an acellular mutated vaccine did not affect liver drug metabolism, indicating a role of the whole bacterial cell in this side effect. All the cellular vaccines studied induced high serum interleukin-6 levels; on the other hand, the acellular mutated vaccine induced very low interleukin-6 levels, indicating that the whole bacterial cell is also important for interleukin-6 induction. All vaccines studied were very poor tumor necrosis factor inducers.

Ciliary neurotrophic factor (CNTF) induces serum amyloid A, hypoglycaemia and anorexia, and potentiates IL-1 induced corticosterone and IL-6 production in mice.

Ciliary neurotrophic factor (CNTF) supports the survival of ciliary ganglion neurons and was shown to induce the synthesis of acute-phase proteins and fever. We studied the effect of CNTF, alone or in association with IL-1, on levels of corticosterone (CS), glucose, serum amyloid A (SAA), and IL-6. We also compared the effect of CNTF with that of IL-6, since the gp130 receptor subunit for CNTF is shared with that of IL-6. A single intravenous injection of CNTF induced hypoglycaemia and SAA and potentiated IL-1-induced CS and IL-6. Chronic CNTF, but not IL-6, resulted in decreased food intake and body weight up to days 6-7. After this time, body weight and food intake recovered even if CNTF treatment was continued, indicating that a phenomenon of tolerance occurred. Finally, CNTF (unlike IL-1) was not toxic in adrenalectomized mice. Therefore the similarities of CNTF activities with those of other cytokines, particularly IL-6, might go beyond the activation of the same receptor-signal transduction pathway of IL-6.