Small Amounts Of Nitric Oxide Research Articles

P407 hydrogel loaded with nitric oxide microbubbles promotes angiogenesis and functional improvement in testicular transplantation.

Prepubertal male patients with cancer have decreased fertility after treatment, but there are currently no suitable means for fertility rescue. Testicular transplantation seems to be a promising treatment. The short-term insufficiency of blood supply after transplantation is the key problem that needs to be solved. In this research, nitric oxide (NO), a gas and small molecule transmitter with the effect of promoting angiogenesis, acted at the site of testicular transplantation. Herein, poloxamer-407 (P407) and lipid microbubble materials served as transport carriers for NO and helped NO to function at the transplant site. P407 hydrogel loaded with NO microbubbles (PNO) slowly released NO in vitro. The three-dimensional space of the hydrogel provided a stable environment for NO microbubbles, which is conducive to the continuous release of NO. In this study, 25% PNO (w/v) was selected, and the gelling temperature was 19.47 °C. The gelling efficiency was relatively high at body temperature. Rheological experiments showed that PNO, at this concentration, had stable mechanical properties. The results from in vivo experiments demonstrated that testicular grafts in the PNO group exhibited a notably accelerated blood flow recovery compared to the other groups. Additionally, the PNO group displayed a significant improvement in reproductive function recovery. In conclusion, PNO exhibited slow release of NO, and a small amount of NO promoted angiogenesis in testicular grafts and restored reproductive function.

GENE POLYMORPHISM AS A PREDICTOR DEVELOPMENT OF THE CHILDREN DISEASE

Neonates suffering from severe birth asphyxia may develop hypoxic ischemic encephalopathy and in some cases to permanent neurological damage. Around 20 – 50% of neonates with birth asphyxia who have hypoxic ischemic encephalopathy symptoms die in the neonatal period. Our study aims to clarify the role and impact of gene polymorphisms on the occurrence of hypoxic-ischemic encephalopathy. Hypoxic-ischaemic encephalopathy is a common cause of death and disability in newborns. It causes long-term or permanent damage, such as cerebral palsy, epilepsy, and certain forms of mental retardation. Autoregulation may be impaired during various neonatal disease and states including prematurity, hypoxic-ischemic encephalopathy, intraventricular haemorrhage, congenital cardiac disease, and infants requiring extracorporeal membrane oxygenation. As infants are especially sensitive to changes in cerebral blood flow, both hypoperfusion and hyperperfusion can cause significant neurologic injury. All of these conditions impair cerebral blood flow, leading to ischemia and hypoxia and trigger a cascade of deleterious biochemical events that seriously and permanently injure the brain. The nitric oxide produced by NOS3 plays a critical role in maintaining cerebral blood circulation and preventing neuronal injury. Mild exposure to ischemia activates enzyme NOS3 and produces small amounts of nitric oxide with subsequent relaxation of blood vessels and vasodilatation. Genetic factors could also affect the processes of autoregulation and destruction following hypoxic-ischemic injury. An underlying genetic predisposition that increases or decreases the degree of injury after a prenatal hypoxic ischemic event may affect the clinical course and prognosis. Except of therapeutic hypothermia, which currently is the only treatment available, a considerable number of newborns still have adverse outcomes. Therefore, there is a pressing need for novel better prognostic and diagnostic tools to improve outcome.

Photolytic or Oxidative Fragmentation of Trityl Diazeniumdiolate (O2N2CPh3–): Evidence for Both C–N and N–N Bond Cleavage

Exposure of [K(18-crown-6)(THF)2][CPh3] (THF = tetrahydrofuran; Ph = phenyl) to an atmosphere of nitric oxide (NO) cleanly generates [K(18-crown-6)][O2N2CPh3] (1) in excellent yields. A subsequent reaction of [ZnCl2(THF)2] with 3 equiv of 1 affords the C-diazeniumdiolate complex [K(18-crown-6)][Zn(O2N2CPh3)3] (2). Both 1 and 2 were characterized by 1H and 13C{1H} NMR spectroscopy, and their structures were confirmed by X-ray crystallography. Photolysis of 2 using 371 nm light resulted in the formation of three trityl-containing products, namely, Ph3CH, 9-phenylfluorene, and Ph3CN(H)OCPh3 (3). In addition, we detected nitrous oxide (N2O), as well as small amounts of NO in the reaction mixture. In contrast, oxidation of 2 with 1.2 equiv of [Ag(MeCN)4][PF6] resulted in the formation of O(CPh3)2 as the major trityl-containing product; N2O was also detected in the reaction mixture, but NO was not apparently formed in this case. The observation of these fragmentation products indicates that the [O2N2CPh3]- ligand is susceptible to both C-N bond and N-N bond cleavage. Moreover, the different product distributions suggest that [O2N2CPh3]- is susceptible to different modes of fragmentation.

The role of nitric oxide in peptic ulcer: a narrative review.

Peptic ulcer refers to the inflammatory response and necrotic lesions of the mucosa under the action of various pathogenic factors, which goes deeply into the mucosal muscle layer and often occurs to the gastrointestinal mucosa related to gastric acid secretion, among which the stomach and duodenum are the most common. The clinical manifestations include slow onset, prolonged course and weekly upper abdominal pain. Nitric oxide (NO) is an intracellular and intercellular signaling molecule that plays an important role in many physiological and pathological processes. Studies have found that a small amount of NO produced in vivo plays a role in many physiological homeostasis, such as regulating blood pressure, platelet aggregation, nitrogenization of hemoglobin, and regulating proliferation and differentiation of stem cells. However, under the action of some cytokines and oxidative stress, intracellular NO synthase will catalyze the synthesis of large amounts of NO and participate in the inflammatory response, causing beneficial or harmful effect on the body. Numerous basic studies have focused on the relationship between NO and peptic ulcer. The purpose of this review is to summarize the role of NO in peptic ulcer and its possible mechanism.

Kinetic modeling of nitrous oxide decomposition on Fe-ZSM-5 in the presence of nitric oxide based on parameters obtained from first-principles calculations

A variety of experiments for the N2O decomposition over Fe-ZSM-5 catalysts have been simulated in the presence and absence of small amounts of nitric oxide and water vapor.

Nitric oxide formation in lean, methane-air stagnation flames at supra-atmospheric pressures

Increasingly stringent regulations are imposed on nitric oxide (NO) due to its numerous, direct and indirect, deleterious effects on human health and the environment. A better control of the post-flame temperature field to contain the thermal (Zel’dovich) route resulted in significant reductions of engine emissions. An improved knowledge of the chemistry and rate of the secondary prompt, NNH, and N2O pathways is now required to decrease nitric oxide emissions further. For this effort, NO laser-induced fluorescence (LIF) measurements are presented for lean (ϕ=0.7), jet-wall, stagnation, premixed flames at pressures of 2, 4, and 8 atm. For all cases, the NO-LIF signal increases rapidly through the flame front, and relatively slowly in the post-flame region where the temperature is too low to sustain the thermal pathway. Nitric oxide mole fractions are inferred from the measurements and show that the pressure has a very weak, monotonic, adverse effect on NO formation. Reaction pathway analyses are applied to flame simulations performed with a thermochemical model capturing the general trends of the data to assess the contribution of each NO formation route. For all cases, the N2O pathway, which proceeds mostly in the flame front region, dominates. This route produces slightly larger amounts of NO at higher pressures, but the variation appears very limited when considering the termolecular nature of its initiation reaction. The thermal route is predicted to progress slowly in the post-flame region, which causes a shallow increase in the NO mole fraction. The NNH and prompt pathways generate small amounts of NO, and their contributions reduce with the pressure. Overall, the thermochemical model predicts that the formation of NO is relatively unaffected by the pressure, which is consistent with the experiments. The experimental dataset reported in this work is made available for the development of thermochemical models.

Structure and Reactivity of sol–gel V/SiO2 Catalysts for the Direct Conversion of Methane to Formaldehyde

Vanadium oxide-silica catalysts prepared by the sol–gel method were characterized by different techniques (nitrogen adsorption–desorption isotherms, scanning electron microscopy, X-ray diffraction, temperature-programmed reduction, Raman spectroscopy and X-ray photoelectron spectroscopy) and applied in the direct conversion of methane to C1 oxygenates. The addition of a small amount of nitric oxide to the reaction mixture reduced the energy barrier for H-abstraction, increasing the methane conversion and formaldehyde yield. Correlations between the characterization and activity results indicate that the reaction occurs on tetrahedrally coordinated vanadium sites, as the maximum formaldehyde yield was found for the catalyst with a vanadium content of 1.5 wt%, which has a high surface density of well-dispersed tetrahedrally coordinated monomeric or slightly oligomerized VO4 species. On the other hand, a high space velocity and CH4:O2 ratio decrease the subsequent oxidation to carbon oxides, increasing oxygenate formation.

Photometric Detection of Nitric Oxide Using a Dissolved Iron(III) Corrole as a Sensitizer.

The potential of an iron(III) corrole complex for use in the detection of nitric oxide (NO) was investigated. The reversible conversion of an dissolved iron(III) corrole to its corresponding nitrosyl complex using gaseous nitric oxide was monitored by UV/Vis spectroscopy. The spectral differences between both coordination compounds were used to determine photometrically small amounts of nitric oxide in the sub-parts-per-million range. The spectral changes due to NO binding were assigned to charge-transfer transitions arising upon NO coordination and were analyzed in detail with support from quantum chemical calculations. Finally, films of the iron(III) corrole were deposited on quartz glass. Thus, the great potential of iron(III) corroles for the development of advanced, highly sensitive and low-energy-consuming photonic sensing devices was demonstrated.

Inducible Nitric Oxide Synthase as a Possible Target in Hypertension

Nitric oxide (NO) is an important vasodilator produced by vascular endothelium. Its enzymatic formation is derived from three different synthases: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) synthases. While relatively small amounts of NO produced by eNOS are important to cardiovascular homeostasis, high NO levels produced associated with iNOS activity may have detrimental consequences to the cardiovascular system and contribute to hypertension. In this article, we reviewed current literature and found mounting evidence indicating that increased iNOS expression and activity contribute to the pathogenesis of hypertension and its complications. Excessive amounts of NO produced by iNOS up-regulation can react with superoxide anions forming peroxynitrite, thereby promoting nitrosative stress and endothelial dysfunction. In addition, abnormal iNOS activity can up-regulate arginase activity, allowing it to compete with eNOS for L-arginine, thereby resulting in reduced NO bioavailability. This may also lead to eNOS uncoupling with enhanced production of superoxide anions instead of NO. All these alterations mediated by iNOS apparently contribute to hypertension and its complications. We also reviewed current evidence showing the effects of iNOS inhibitors on different animal models of hypertension. iNOS inhibition apparently exerts antihypertensive effects, decreases oxidative and nitrosative stress, and improves vascular function. Together, these studies highlight the possibility that iNOS is a potential pharmacological target in hypertension.

Characterization of Local Vascular Effects of the Nitric Oxide Inhibitor NG‐Monomethyl‐L‐Arginine on Dorsal Hand Veins

Infusion of NG-monomethyl-L-arginine (L-NMMA; 6.4 µmol/min) into hand veins can cause a 20% increase in vein size in specific subjects. This study explored potential underlying mechanisms in healthy male participants. Ten healthy male participants received in phenylephrine (PE)-preconstricted veins a dose-response curve (DRC) to L-NMMA (0.2-6.4 µmol/min) without and with coinfusion of the endothelium-dependent dilator histamine, a DRC to L-arginine with and without coinfusion of L-NMMA, a DRC to NG-monomethyl-D-arginine (D-NMMA), and a DRC to L-NMMA in prostaglandin F(2α)-(PGF(2α))-preconstricted veins. Participants were classified as L-NMMA responders (R) and nonresponders (NR). Infusion of L-NMMA resulted in a maximum venodilation of 38% ± 11% (R) versus 10% ± 5% (NR; P = .005). In PGF(2α)-preconstricted veins, L-NMMA caused venodilation to 26% ± 34% (NS) in responders. Results suggest that endothelial nitric oxide synthase-mediated formation of nitric oxide (NO) from L-NMMA in doses >3.2 µmol/min and continuous PE-induced α-adrenergic stimulation resulting in release of very small amounts of NO from L-NMMA contribute to the observed L-NMMA-induced increase in vein size. Venous reactivity to L-NMMA resulting in a phenotype as R or NR is most likely genetically predetermined, which requires further study.

Detection of nitroxyl (HNO) by membrane inlet mass spectrometry

Membrane inlet (or introduction) mass spectrometry (MIMS) was used to detect nitroxyl (HNO) in aqueous solution for the first time. The common HNO donors Angeli's salt (AS) and Piloty's acid (PA), along with a newly developed donor, 2-bromo-N-hydroxybenzenesulfonamide (2-bromo-Piloty's acid, 2BrPA), were examined by this technique. MIMS experiments revealed that under physiological conditions 2BrPA is an essentially pure HNO donor, but AS produces a small amount of nitric oxide (NO). In addition, MIMS experiments also confirmed that PA is susceptible to oxidation and NO production, but that 2BrPA is not as prone to oxidation.

Effect of NO on extinction and re-ignition of vortex-perturbed hydrogen flames

The catalytic effect of nitric oxide (NO) on the dynamics of extinction and re-ignition of a vortex-perturbed non-premixed hydrogen–air flame is studied in a counterflow burner. A diffusion flame is established with counterflowing streams of nitrogen-diluted hydrogen at ambient temperature and air heated to a range of temperatures that brackets the auto-ignition temperature. Localized extinction is induced by impulsively driving a fuel-side toroidal vortex into the steady flame, and the recovery of the extinguished region is monitored by planar laser-induced fluorescence (PLIF) of the hydroxyl radical (OH). The dynamics of flame recovery depend on the air temperature and fuel concentration, and four different recovery modes are identified. These modes involve combinations of edge-flame propagation and the expansion of an auto-ignition kernel that forms within the extinguished region. The addition of a small amount of NO significantly alters the re-ignition process by shifting the balance between chain-termination and chain-propagation reactions to enhance auto-ignition. The ignition enhancement by this catalytic effect causes a shift in the conditions that govern the recovery modes. In addition, the effects of NO concentration and vortex strength on the flame recovery are examined. Direct numerical simulations of the flame–vortex interaction with and without NO doping show how the small amount of OH produced by NO-catalyzed reactions has a significant impact on the development of an auto-ignition kernel. This joint experimental and numerical study provides detailed insight into the interaction between transient flows and ignition processes.

Endocannabinoid System and Nitric Oxide are Involved in the Deleterious Effects of Lipopolysaccharide on Murine Decidua

Endocannabinoids are an important family of lipid-signaling molecules that are widely distributed in mammalian tissues and anandamide (AEA) was the first member identified. The uterus contains the highest concentrations of AEA yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. Previous results from our laboratory have shown that AEA modulated NO synthesis in rat placenta. The production of small amounts of nitric oxide regulates various physiological reproductive processes such as implantation, decidualization and myometrial relaxation. But in an inflammatory setting such as sepsis, NO is produced in big amounts and has toxic effects as it is a free radical. The results presented in this study indicate that LPS-induced NO synthesis and tissue damage were mediated by AEA. Decidual LPS-induced NO production was abrogated either by co-incubation with CB1 (AM251) or CB2 (SR144528) antagonists which suggests that both receptors could be mediating this effect. On the other hand, LPS-induced tissue damage and this deleterious effect was partially abrogated by incubating tissue explants with LPS plus CB1 receptor antagonist. Our findings suggest that AEA, probably by increasing NO synthesis, participates in the deleterious effect of LPS in implantation sites. These effects could be involved in pathological reproductive events such as septic abortion.

Anandamide regulates lipopolysaccharide-induced nitric oxide synthesis and tissue damage in the murine uterus

In women, the association between chronic marijuana smoking and early miscarriage has long been known. Anandamide, a major endocannabinoid, mimics some of the psychotropic, hypnotic and analgesic effects of Delta(9)-tetrahydrocannabinol, the psychoactive component of marijuana. The uterus contains the highest concentrations of anandamide yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. The production of small amounts of nitric oxide (NO) regulates various physiological events including implantation and myometrial relaxation, but in an inflammatory setting such as sepsis, NO has toxic effects as it is a free radical. The results presented in this study indicate that anandamide modulates NO production induced by lipopolysaccharide (LPS) in an in-vitro murine model. It was shown that LPS-induced NO synthesis and tissue damage were mediated by anandamide, as a cannabinoid receptor type I antagonist could block the effect of LPS (P < 0.001). This endotoxin inhibited anandamide uterine degradation (P < 0.05) and increased the expression of one of its synthesizing enzymes (P < 0.05). Contrary to the known anti-inflammatory and protective effects, in this model anandamide seems to act as a pro-inflammatory molecule modulating the production of NO induced by LPS. This proinflammatory effect of anandamide may be implicated in pathological reproductive events such as septic abortion.

Nasal mucosal expression of nitric oxide synthases in patients with allergic rhinitis and its relation to asthma

Nitric oxide (NO) has contradictory roles in the pathophysiology of allergic inflammation in both allergic rhinitis (AR) and asthma. Small amounts of NO produced by constitutive NO synthase (NOS) is anti-inflammatory, whereas large amounts produced by inducible NOS (iNOS) are proinflammatory. To investigate the difference in constitutive endothelial NOS (eNOS) and iNOS expression in nonallergic and allergic mucosa and the possible relation of this to the coexistence of asthma in seasonal AR. Seventeen patients (10 women and 7 men) with seasonal AR and 9 nonallergic patients (5 women and 4 men) with nasal septum deviation were enrolled. Inferior turbinate nasal biopsy specimens were obtained in all. Levels of eNOS and iNOS expressed as immunohistochemical scores (HSCOREs) were determined immunohistochemically from the specimens. The mean +/- SD HSCOREs for eNOS in patients with seasonal AR were not significantly different from those of the nonallergic controls (1.85 +/- 0.78 vs 1.63 +/- 0.54; P = .12). On the other hand, the mean +/- SD HSCOREs for iNOS were significantly higher in patients with seasonal AR (1.75 +/- 0.75 vs 0.71 +/- 0.6; P = .004). Furthermore, although eNOS expression was not different between seasonal AR patients with and without asthma, the mean +/- SD HSCOREs for iNOS were significantly higher in the patients with asthma (1.93 +/- 0.78 vs 1.65 +/- 0.55; P = .01). Increased expression of iNOS might have a role in the development of allergic inflammation in upper and lower airways and in comorbidity of AR and asthma.

Exhaled NO and iNOS expression in sputum cells of healthy, obese and OSA subjects

Obstructive sleep apnoea (OSA) is associated with airways inflammation; a key role in this regard seems to be played by nitric oxide (NO). The aim of this study was to measure exhaled NO and expression of its enzyme, the inducible nitric oxide synthase (iNOS) in cells of induced sputum in OSA patients and in obese subjects without sleep apnoea and to correlate these inflammatory markers with severity of OSA. We enrolled 18 obese patients with OSA (10 men, age 48.2 +/- 8.4 years), 15 obese patients without OSA (eight men, age 52.8 +/- 11 years) and 10 healthy subjects (five men, age 42 +/- 4 years). Exhaled NO was measured using a chemiluminescence analyser; iNOS expression was measured in the sputum cells by immunocytochemistry. Exhaled NO resulted significantly increased in OSA and in obese patients (23.1 +/- 2.1 and 17.9 +/- 2.1 p.p.b.) than in healthy subjects (7.2 +/- 0.6 p.p.b.; P < 0.001). OSA and obese patients showed a higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to healthy subjects. In addition, OSA and obese patients showed higher iNOS expression in neutrophils and in macrophages with respect to healthy subjects. A positive correlation between exhaled NO, iNOS expression and AHI was observed. These data confirm the presence of airway inflammation in OSA and in obese patients, and suggest the possible role for NO and iNOS expression in neutrophils of the induced sputum as noninvasive markers to identify and monitor the airway inflammation in these subjects.

Role of Blood- and Tissue-Associated Inducible Nitric-Oxide Synthase in Colonic Inflammation

There is evidence that inducible nitric-oxide synthase (iNOS)-derived NO contributes to the pathophysiology of intestinal inflammation. The aims of this study were to assess the role of iNOS in the development of dextran sodium sulfate (DSS)-induced colonic inflammation and to define the contribution of tissue-specific iNOS expression to this inflammatory response. Study groups included: 1) wild-type (WT) mice; 2) WT=>WT bone marrow chimeras with normal iNOS function; 3) WT=>iNOS-/- chimeras (with functional blood cell iNOS, but iNOS-deficient tissue); 4) iNOS-/-=>WT chimeras (with iNOS-deficient blood cells, but normal tissue iNOS activity); and 5) iNOS-deficient mice. In WT mice and WT=>WT chimeras, DSS-induced colonic inflammation was characterized by bloody diarrhea and a high disease activity index. However, WT=>iNOS-/- and iNOS-/-=>WT chimeras and iNOS-/- mice exhibited an attenuated disease activity index, with parallel changes in histopathology. Colonic myeloperoxidase (MPO) was comparably elevated in DSS-treated WT mice (30.1+/-1.7) and WT=>WT chimeras (29.0+/-1), whereas MPO was significantly reduced in iNOS-/- mice and iNOS-/-=>WT chimeras (9.5+/-1.7 and 15.6+/-2.2, respectively). WT=>iNOS-/- chimeras exhibited the lowest MPO activity (3.7+/-0.6). Our findings implicate both blood cell- and tissue-derived iNOS in DSS-induced colonic inflammation, with tissue-associated iNOS making a larger contribution to the recruitment of inflammatory cells.

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.

Laser-based temperature imaging close to surfaces with toluene and NO-LIF

Two novel techniques based on Laser-Induced Fluorescence (LIF) were applied to measure gas-phase temperature distributions in boundary layers close to wall surfaces. Singleline toluene-LIF thermometry was used to image temperature in a nitrogen gas flow above a heated wall. The nitrogen gas flow was doped with evaporated toluene. When excited at 266 nm, the toluene LIF-signal shows an exponential dependence on temperature. This behavior was used to calculate absolute temperatures from LIF images after calibration at known conditions. The second technique, multi-line NO-LIF thermometry was applied to image temperature in the quenching boundary layer close to a metal wall located on a flat flame burner. A small amount of nitric oxide was mixed into the air/methane mixture. NO molecules were excited in the A-X (0, 0)-band at 225 nm. NO-LIF excitation spectra were acquired by tuning the excimer laser wavelength and recording the NO LIF-signal with an ICCD camera. Absolute temperatures were calculated for every pixel by fitting simulated excitation spectra to the experimental data. Temperature distributions close to the wall surface were measured at two different flow-rate conditions. A high nominal spatial resolution of 0.016 mm/pixel in direction perpendicular to the wall was reached. Wall surface temperatures were recorded simultaneously by embedded thermocouples and compared with gas-phase temperature near the wall surface.

IFN‐γ enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.