Prevention of murine cardiac allograft rejection with gallium nitrate. Comparison with anti-CD4 monoclonal antibody.

Gallium nitrate (GN) was evaluated for its ability to interfere with a cute rejection of DBA/2-->C57BL/6 heterotopic cardiac allografts, in comparison with the depleting anti-CD4 mAb, GK1.5. The administration of GN for 30 days (s.c. 30 mg/kg elemental gallium on days 0 and 3, 10 mg/kg every third day) resulted in >60-day graft survival in 78% (25 of 32) of the graft recipients, whereas 2 perioperative injections of anti-CD4 monoclonal antibody (mAb) resulted in >60-day graft survival in 58% (24 of 41) of the graft recipients. Serum gallium levels peaked at about 2000 ng/ml after 2-3 weeks of treatment and decreased to about 300 ng/ml by day 60, a level that was maintained for at least 30 more days. During the early posttransplant period, 25% of GN-treated grafts, but not anti-CD4 mAb-treated grafts, exhibited an unusual, transient reduction in graft impulse strength, suggesting a transient rejection response. Macroscopically, the long-surviving (>60 days) grafts from either treatment group exhibited none of the features of rejecting allografts. Histologically, they exhibited minor edema and rare epicardial inflammation but no tissue necrosis. However, there were vascular changes in allografts from GN-treated mice, including altered endothelial morphology, associated with moderate intimal hyperplasia and mild perivascular leukocytic infiltration. Allografts from anti-CD4 mAb-treated mice exhibited prominent neointimal hyperplasia associated with endothelial morphologic changes and prominent vascular and perivascular leukocytic infiltration. In general, both GN and anti-CD4 mAb promoted long-term allograft survival, but these allografts displayed the histopathologic signs of ongoing inflammation and chronic allograft rejection.

Gallium nitrate suppresses the production of nitric oxide and liver damage in a murine model of LPS-induced septic shock.

The efficacy of gallium (Ga) nitrate was examined in a murine model of sepsis. Male Balb/c mice (6-8 weeks) were randomized into 3 groups: 1) vehicle-treated controls 2) mice with sepsis induced by treatment with 0.3 mg i.v. of Propionibacterium acnes followed one week later by 0.01 microg lipopolysaccharide (LPS) and 10 mg of D-galactosamine (GalN) 3) mice with sepsis injected with 45 mg/kg s.c. of gallium nitrate (calculated as elemental Ga) 24 hours prior to LPS/GalN. Two hours after LPS/GalN or vehicle, plasma concentrations of tumor necrosis factor (TNF-alpha) in groups 1, 2 and 3 were 54+/-31 (n=6), 21,390+/-5139 (n=4), and 21,909+/-943 (n=5) pg/ml, respectively. After 6 hours, plasma concentrations of gamma interferon (IFN-gamma) were <10 (n=8), 4771+/-1078 (n=6), and 1622+/-531 (n=15) pg/ml, respectively, and of nitrate/nitrite (products of nitric oxide) were 64+/-8 (n=7), 146+/-18 (n=8), and 57+/-8 (n=15) microM. At 18 hours, serum chemistries were; SGOT 171+/-46 (n=13), 10,986+/-3062 (n=7), and 1078+/-549 (n=8) IU/L; SGPT 165+/-59, 17,214+/-4340, and 2088+/-1097 IU/L; and total bilirubin 0.2+/-0.0, 0.9+/-0.4, and 0.2+/-0.0 mg/dl for groups 1, 2, and 3 respectively. Blinded histologic evaluation of livers at 18 hours revealed inflammatory infiltrate scores (x [range], 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe) of 0.1 [0-1] (n=8), 3.0 [2-4] (n=15), and 2.0 [0-3] (n=10), and necrosis scores of 0.0, 2.8 [0-4], and 0.9 [0-4]. Although Ga did not affect production of TNF-alpha, it ameliorated hepatocellular injury and protected against necrosis. Based on this model of sepsis, Ga may have a role in treating the human disease.

Prenatal smokeless tobacco effects on the rat fetus.

The objective of this study was to examine the effect of an aqueous extract of smokeless tobacco (ST) on the development and bone ossification of the Sprague Dawley rat fetus at known nicotine blood levels. Dams were intubated with the ST extract three times daily on gestational days (GD) 6-18 with one of the following: ST equivalent to either 1.33 mg nicotine/kg body weight (STD-1), 6 mg nicotine/kg body weight (STD-2), or equal amounts of distilled water (sham-treated controls). Parallel groups of rats were used for nicotine-blood-level determinations. Dams were killed on GD 19, fetuses and placentas were weighed, and resorptions, deaths, and/or malformations were noted. Two thirds of the fetuses were further examined by Wilson's method, and the remaining one third was stained and cleared for skeletal examinations. Mean plasma nicotine levels, determined in a parallel group of nonpregnant/pregnant rats, were 220.4/283.3 ng/ml in the STD-1 group and 869.1/846.3 ng/ml in the STD-2 group. At these ST dosages, weight gain of dams was reduced in comparison with sham-treated controls (P < .05), but fetal weights were reduced in the STD-2 group only. Placental weights, litter size, resorptions, deaths, and malformations were not significantly affected. Skeletal examinations revealed several dose-related differences between the ST-treated and sham-treated control groups. In the STD-1 group, reductions in ossification were seen in the nasal and femur width measurements only. In the STD-2 group, reductions in ossification were seen in femur length and width, in the number of ossification centers in the forelimb, in the maxillary, mandibular, and nasal bone measurements. We conclude that under these experimental conditions the effects of ST at the low dose are minimal, whereas the high ST dose resulted in significant growth retardation and decreased ossification levels (P < .05).

Alcohol and smokeless tobacco effects on the CD-1 mouse fetus.

Tobacco products and alcohol are commonly used as nonmedicinal drugs by pregnant women, and both are known to cause various effects on the fetus and the newborn. The objective of this study was to examine the fetal effects of both drugs when administered individually and simultaneously to pregnant CD-1 mice at moderate dosages. Specifically, we wanted to determine whether or not the effect on the fetus of these two biologically active substances was additive, ameliorative, or synergistic. A total of 65 CD-1 dams were divided among four groups receiving either ST equivalent to 8 mg/kg nicotine, ethanol (ETOH) 1.8 g/kg, a combination of ST+ETOH in the same dosages, or D-glucose (controls and ST alone) to supply calories equivalent to the dose of ethanol. Mice were dosed three times per day on gestational days 6-15. On gestational day 17 all dams were killed, fetal and placental weights recorded, and the number of resorbed, dead, and malformed fetuses noted. The mean maternal plasma drug levels were: nicotine-321 ng/ml and ethanol-0.105 g%. No significant differences were observed in maternal weight gain, litter size, or in the incidence of resorptions, deaths and/or malformations. Fetal weights were reduced in all three treatment groups (P less than 0.05), with the greatest reduction (13% decrease) recorded in the ST group, followed by a 9% decrease in the ETOH group, and a 7% decrease in the ST+ETOH group. Placentas of the ST group weighed significantly less (P less than 0.05) than controls. Ossification of the fetal skeleton, observed in ten sites, was affected to the greatest extent in the ST group, followed by the ETOH and ST+ETOH groups. Craniofacial measurements were significantly affected (P less than 0.05) in all three treatment groups, compared to controls. We conclude that under these experimental conditions, in terms of fetal growth and ossification, ST had the greatest effect, followed by ETOH and ST+ETOH. The interaction of ST+ETOH was neither additive, synergistic, nor ameliorative.

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.

An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages.

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.

Endogenous nitric oxide inhibits endothelin-1-induced chloride secretion in guinea pig colon.

Segments of guinea pig distal colon, stripped of the external muscle layers, were set up in flux chambers for measurement of short-circuit current (Isc) indicative of active, electrogenic ion transport. During neural blockade with tetrodotoxin, the nitric oxide scavenger, hemoglobin, and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA), reduced Isc. The reduction in Isc in response to hemoglobin was reversed by L-arginine and blockers of chloride secretion, including bumetanide and diphenylamine-2-carboxylic acid, but not by the potassium channel blockers, barium and tetraethylammonium, nor by amiloride, an epithelial sodium channel blocker. The hemoglobin-induced reduction in Isc was not affected by blockade of prostaglandin synthesis with piroxicam. During neural blockade, the nitric oxide donors, sodium nitroprusside and NONOate, increased Isc which was abolished by piroxicam. Endothelin-1 (ET-1) also evoked an increase in Isc that was unaffected by amiloride and was inhibitable by bumetanide, chloride-free solutions, tetrodotoxin, piroxicam, and the ETA receptor antagonist, BQ123. The ETB receptor agonist, [Ala1,3,11,15]-endothelin-1, had no appreciable effect on Isc. Hemoglobin and L-NNA enhanced the ET-1-induced Isc response by about twofold without affecting prostaglandin E2 release or its secretory response. The results suggest that endogenous nitric oxide stimulates a low level of chloride secretion that is independent of prostaglandins, unlike nitric oxide donors which increase chloride secretion by releasing prostaglandins. In addition, endogenous nitric oxide suppresses ET-1-evoked chloride secretion by mechanisms that are unrelated to the release of prostaglandin E2 or its ability to stimulate epithelial cells. Endogenous nitric oxide may play an important role in modulating chloride secretion during ischemic challenge when endothelin levels are high.

In vivo disposition and metabolism by liver and enterocyte microsomes of the antitubercular drug rifabutin in rats.

The in vivo disposition and in vitro metabolism of rifabutin, a new spiropiperidylrifamycin, were studied in rats and in microsomes from rat liver and enterocytes, respectively. After i.v. doses of 1,5, 10 and 25 mg/kg the systemic clearance was 0.7 to 1.0 liters/hr/kg; the volume of distribution was 4.4 liters/kg for the 1 mg/kg dose and 7.4 to 7.7 liters/kg for the 5 to 25 mg/kg doses, and the half-life ranged from 4.4 to 9.1 hr. Urinary and fecal excretion over 0 to 96 hr after i.v. administration of 25 mg/kg [14C]rifabutin accounted for 40.1 and 52.2% of the dose, respectively. Exteriorization of the bile duct showed that approximately 24% of the dose was eliminated in bile, > or = 98% as metabolites. Bioavailability after oral administration of 25 and 1 mg/kg rifabutin was > 90% and 44%, respectively, suggesting significant first-pass metabolism of the lower dose. Concentrations of rifabutin in gastric juice were 10 to 17 times higher than in blood, indicating extensive secretion into the stomach. Experiments with the isolated small intestinal loop demonstrated direct exsorption of the drug into the lumen. The rate of rifabutin metabolism by enterocyte microsomes was > 10 times higher than that by liver microsomes, i.e., 84 and 8 pmol/min/mg protein, respectively. Biotransformation of rifabutin in vivo and in vitro was markedly induced by dexamethasone and inhibited by erythromycin, suggesting that CYP3A is involved in the metabolism of rifabutin. Several metabolites, including 20-OH-rifabutin and 27-O-demethyl-rifabutin, isolated from urine and microsomes were identified by mass spectrometry and nuclear magnetic resonance spectroscopy.

The effects of ziprasidone on steady-state lithium levels and renal clearance of lithium.

AIMS: To assess the potential of ziprasidone to alter the renal clearance and steady-state serum levels of lithium. METHODS: Healthy subjects who had stable serum lithium levels during the first 7 days of treatment with lithium 900 mg day(-1), given as two divided daily doses, were randomized to receive concomitant treatment with either ziprasidone, 40 mg day(-1), given as two divided daily doses, on days 9-11 followed by 80 mg day(-1), given as two divided daily doses on days 12-15 (n = 12), or placebo twice daily (n = 13). Ziprasidone or placebo was administered 2 h before each dose of lithium. RESULTS: Ziprasidone administration was associated with a 0.07 mmol l(-1) (13%) mean increase in steady-state serum lithium levels compared with a mean increase of 0.06 mmol l(-1) (10%) with placebo. Mean renal clearance of lithium decreased by 0.09 l h(-1) (5%) in the ziprasidone group and by 0.14 l h(-1) (9%) in the placebo group. None of these differences between the two groups was statistically or clinically significant. CONCLUSIONS: Ziprasidone does not alter steady-state serum lithium concentrations or renal clearance of lithium.

Effect of gallium nitrate on experimental autoimmune uveitis.

Gallium nitrate (GN) has been shown to inhibit T cell-mediated inflammatory disease. The purpose of our study was to test the effect of gallium nitrate (GN) on experimental autoimmune uveitis (EAU). Experimental autoimmune uveitis was induced in male Lewis rats immunized with retinal S-antigen. Rats received subcutaneous injections of GN or saline one day prior to immunization and 1, 4, 7, 10, 13, 16, and 19 days after immunization. Ocular inflammation was graded clinically and histologically by masked observers, and in vitro assays of cell-mediated and humoral immunity were performed. GN significantly inhibited the development of EAU graded clinically (P = 0.001) and histologically (P = 0.002). Treatment with GN also resulted in a small (30-41%) decrease in the lymphocyte responses to retinal S-Antigen and a small (12-37%) reduction in antibody production to S-antigen. These data show that GN suppresses the development of EAU, and inhibits both lymphocyte proliferative responses to antigen and antibody production.