House dust mite induces direct airway inflammation in vivo: implications for future disease therapy?

House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma. HDM can evoke a direct, nonallergic inflammatory reaction in vitro. We aimed to determine whether this apparent nonallergic, inflammatory response can be observed in a more complex in vivo setting. Vehicle, Alum or HDM (Dermatophagoides pteronyssinus 5 microg, i.p. with Alum) sensitised Brown-Norway rats were challenged intratracheally with vehicle (saline), HDM (Der p 10 microg) or heat-inactivated HDM on day 21. Lung function changes and the associated inflammatory response were evaluated. Tissue and bronchoalveolar lavage from Alum sensitised Der p challenged animals exhibited strong eosinophilia and neutrophilia associated with an early release of pro-inflammatory cytokines (interleukin-13 and 1beta, eotaxin and thymus and activation-regulated chemokine). This response was not attenuated by removal of HDM-associated protease activity. Interestingly, the vehicle sensitised group (no Alum) lacked this inflammatory response. HDM allergen evokes nonallergic airways inflammation with an inflammatory profile similar to that of the asthmatic airway. This response, independent of the protease activity of the HDM extract, appeared to be linked to prior administration of the adjuvant Alum and the subsequent increase in total immunoglobulin E. This finding could have important implications in the development of future asthma therapies.

Nitric oxide as a signal in blood vessels.

In the five years since the discovery that nitric oxide is produced as a signal in blood vessels, a great deal has been discovered about the processes involved. This article reviews current knowledge about the vascular cell synthesis, effects and subsequent destruction of this messenger molecule.

Selective iNOS inhibitor 1400W enhances anti-catabolic IL-10 and reduces destructive MMP-10 in OA cartilage. Survey of the effects of 1400W on inflammatory mediators produced by OA cartilage as detected by protein antibody array.

OBJECTIVES: In osteoarthritis (OA), the balance between catabolic and anabolic mediators and their regulators in cartilage is disturbed. Proinflammatory cytokine interleukin-1 (IL-1) plays a central role in cartilage destruction and nitric oxide (NO) mediates many of its destructive effects. In the present study, we investigated the secretion of 40 mediators related to inflammation or cartilage degradation by OA cartilage samples with a protein antibody array. The effects of IL-1 and a selective iNOS-inhibitor 1400W on the mediator release were also studied. METHODS: Cartilage tissue was obtained from the leftover pieces of total knee replacement surgery from OA patients. Protein antibody array was used to measure production of 40 mediators in the culture medium. ELISA was used to confirm the antibody array results. RESULTS: OA cartilage secreted spontaneously 15 out of the 40 measured mediators. IL-1Beta enhanced production of 11 of these inflammatory mediators in OA cartilage along with increased NO production. Treatment with a selective iNOS inhibitor 1400W enhanced the production of IL-10, while the levels of MMP-10 were reduced in IL-1 -treated OA cartilage. CONCLUSION: OA cartilage produces many of the mediators involved in the pathogenesis of OA. The ability of 1400W to enhance levels of anti-catabolic IL-10 and to reduce levels of destructive MMP-10 points to the anti-inflammatory mechanisms that iNOS-inhibitors may have.

Nitric oxide synthase activity in human gynecological cancer.

Nitric oxide is generated by the NO synthases, a family of isoenzymes expressed in a wide range of mammalian cells. In the vascular and nervous systems distinct isoforms generate NO to act as a signal transduction mechanism. The isoform induced by cytokines, on the other hand, provides a sustained release of NO which mediates some cytotoxic and cytostatic effects of the immune system. Solid tumors are a heterogeneous population of cell types, including tumor, vascular, and infiltrating immune cells. Studies in vitro show that NO synthase can be present in many of these cells. However, its presence in situ in solid human tumors has not been reported. In this study, we have investigated NO synthase activity and its cellular localization in malignant and nonmalignant human gynecological tissue. Nitric oxide synthase activity was observed in malignant tissue, was highest (> or = 250 pmol/min/g tissue) in poorly differentiated tumors, and was below detectable levels in normal gynecological tissue. Furthermore, investigations with a polyclonal NO synthase antibody revealed immunoreactivity only in malignant tissue. This was associated with NO synthase activity and localized to tumor cells. Thus NO synthase is present in human gynecological tumors, and its presence seems to correlate inversely with the differentiation of the tumor.

Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: studies with 1400W, a novel inhibitor.

We have investigated the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a novel and highly selective inhibitor for inducible NOS (iNOS), on in vivo growth of solid tumors expressing iNOS. For the EMT6 murine mammary adenocarcinoma, in which iNOS is expressed in the tumor cells, continuous infusion of 1400W for 6 days at 10 or 12 mg/kg(-1)/h(-1) resulted in significant reduction in tumor weight (357 +/- 46 and 466 +/- 70 mg, respectively) compared with that of controls [726 +/- 65 (P < 0.001) and 796 +/- 88 mg (P < 0.02), respectively]. Reduced growth was also observed for a human tumor xenograft (colon adenocarcinoma DLD-1) genetically engineered to express iNOS constitutively and treated for 13 days with 6 mg/kg(-1)/h(-1) 1400W compared with controls (tumor weights 340 +/- 50 and 580 +/- 90 mg, respectively; P < 0.03). Growth of the parental DLD-1 clone was not altered with this treatment compared with that of controls (tumor weights 170 +/- 10 and 240 +/- 50 mg, respectively). Inhibition of iNOS in vivo was confirmed by decreases in plasma nitrite + nitrate concentrations in treated animals compared with that of controls (63-83% decreases for all experiments) and was supported by plasma and tumor concentrations of 1400W that were equivalent and 2.6-4.9 times higher than the EC50 previously reported for iNOS in a tissue assay. For the murine colon adenocarcinoma Colon 38, in which intratumoral macrophages are the predominant source of iNOS and which had high intratumoral arginine concentrations, 1400W treatment had no effect on growth or plasma nitrate + nitrate. Future studies with more potent selective iNOS inhibitors and a wider range of tumors may determine whether iNOS inhibitors could represent a novel approach to the treatment of cancer. These studies confirm that nitric oxide production in tumors plays a role in promoting their growth, rather than a role as a host defense mechanism in inhibiting growth.

Antitumor activity of gamma-interferon in ascitic and solid tumor models of human ovarian cancer.

We have investigated the action of recombinant human gamma-interferon (rHuIFN-gamma) against human ovarian cancer xenografts growing as ascites or as bulky solid i.p. tumors in nude mice. Both forms of the disease responded to i.p. rHuIFN-gamma with significant increases in mouse survival time, and in 2 of 3 ascitic models the mice were cured of peritoneal disease. The activity of rHuIFN-gamma was dose and schedule dependent, and xenografts derived from 3 different patients showed a heterogeneity of response. Peak i.p. levels of rHuIFN-gamma in nude mice bearing multiple i.p. solid tumors were similar to those found in ovarian cancer patients receiving i.p. rHuIFN-gamma, but clearance was more rapid in the mice. Rat gamma-interferon had no antitumor activity at the same doses and schedules although it had some biological activity in the nude mice. Histological examination of treated tumors revealed increased necrosis and loss of cellular organization with large areas of hypocellular epithelial mucin. These changes were preceded by a fall in tumor tryptophan and a rise in tumor kynurenine. We conclude that rHuIFN-gamma has a direct dose related antitumor effect on ovarian cancer xenografts that is preceded by increased metabolism of tryptophan.

No detectable NO synthesis from L-arginine or N(G)-hydroxy-L-arginine in fMLP-stimulated human blood neutrophils despite production of nitrite, nitrate, and citrulline from N(G)-hydroxy-L-arginine.

Nitric oxide (NO) is a well-documented effector molecule in rodent phagocytes but its synthesis in human neutrophils has been controversial. In this study, NO production in human neutrophils activated by chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in the presence of L-arginine (L-Arg) and N(G)-hydroxy-L-arginine (OH-L-Arg), the precursor and intermediate amino acids in NO synthesis, respectively. Incubation of fMLP-activated neutrophils with OH-L-Arg resulted in a production of nitrite, nitrate, and citrulline that was greater than with unstimulated neutrophils but was not inhibited by the NOS inhibitors L-NMMA and L-NIO or the cytochrome P450 inhibitor troleandomycin and was not seen when OH-L-Arg was replaced with L-Arg. This nitrite, nitrate, and citrulline production was not associated with any detectable NO synthesis because no increases in cyclic GMP were observed in the presence of phosphodiesterase inhibitors and in the presence or absence of superoxide dismutase. Moreover, no increases in the formation of the reaction product of NO with superoxide, peroxynitrite, were observed on addition of either OH-L-Arg or L-Arg to activated neutrophils, as assessed either by dihydrorhodamine oxidation or protein nitration. This suggests that, in spite of the production of nitrite, nitrate, and citrulline, commonly used indicators of NO formation, normal human blood neutrophils, are not producing detectable amounts of either NO or peroxynitrite when stimulated with fMLP in the presence of OH-L-Arg.

Pregnancy increases guanosine 3',5'-monophosphate in the myometrium independent of nitric oxide synthesis.

The mechanism for myometrial quiescence during pregnancy is unknown. cGMP plays an integral role in the relaxation of smooth muscle, and nitric oxide (NO) is the most important endogenous activator of soluble guanylate cyclase. The purpose of this study was to determine the effect of gestational age on myometrial cGMP and NO synthase (NOS) activity in the guinea pig. Myometrial cGMP content (measured by RIA) rose slowly until 0.49 (fraction of pregnancy completed) gestation before abruptly increasing to 200 times the non-pregnant control value. It then declined precipitously after 0.87 gestation. Of the known isoenzymes of NOS, the messenger RNAs coding for both endothelial and neuronal NOS could be amplified from the myometrium of pregnant and nonpregnant animals using reverse transcriptase-polymerase chain reaction, but inducible NOS messenger RNA was not found. Myometrial calcium-dependent NOS activity (measured by the conversion of L-[U-14C]arginine to [U-14C]citrulline) declined slowly with advancing gestation (r2 = 0.096; slope = -0.34; P = 0.01), but never differed significantly from the activity in nonpregnant animals [31.1 +/- 11 (term pregnancy) vs. 56.9 +/- 16 (nonpregnant) pmol/min.g; P = NS]. Calcium-independent activity declined shortly after conception, and then rose toward the nonpregnant level (r2 = 0.19; slope = 0.45; P = 0.0009). However, at no time was it significantly different from that in the nonpregnant animal. Pregnancy had no effect on myometrial L-arginine and L-citrulline content. The administration of L-nitro-arginine methyl ester (200 mg/kg) to inhibit NOS dramatically increased blood pressure and reduced fetal renal NOS activity, but had no effect on the myometrial cGMP content. Estradiol (500 micrograms/kg for 5 days) modestly increased cGMP, but in contrast to many tissues in which estradiol increases NOS, it had no effect on myometrial NOS activity. We conclude that pregnancy dramatically increases cGMP by a mechanism independent of NOS. The stimulus remains to be identified. The temporal change in cGMP concentration is consistent with the hypothesis that cGMP mediates myometrial quiescence during pregnancy.

Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases.

The distribution of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase (NOS) was studied in rabbits and in control and endotoxin-treated rats and guinea-pigs. There was a widespread localization of NOS which differed for the two forms of the enzyme and which showed marked differences between species. Endotoxin induced the activity of the Ca(2+)-independent NOS in many tissues and also increased the activity of Ca(2+)-dependent NOS in the rat ileum and caecum. These results demonstrate the differential distribution of NOSs in control and endotoxin-treated animals and emphasize the widespread biological role of nitric oxide (NO).

The formation of nitric oxide donors from peroxynitrite.

1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.

The control of aromatic amino acid catabolism and its relationship to neurotransmitter amine synthesis.

The aromatic amino acids are, inter alia, substrates for the synthesis of important neurotransmitters. Although the factors controlling the synthesis of these transmitters are not fully understood, there is evidence that the concentrations, both relative and absolute, of the precursor amino acids in the blood are of some significance. The article reviews the biochemical pathways involved in tryptophan, phenylalanine, and tyrosine metabolism in liver, brain, and other tissues and discusses (1) the major regulatory events in the maintenance of blood concentrations and (2) the effects of diet, load dosing, hormones, and other circulating substances on the fate of the amino acids and on events in the central nervous system.

Quantitative studies of sulphate conjugation by isolated rat liver cells using [35S]sulphate.

We have developed a simple, rapid and sensitive method for the study of sulphate conjugation in isolated liver cells based on the incorporation of 35S from [35S]sulphate. Excess [35S]sulphate is removed by a barium precipitation procedure, leaving [35S]sulphate conjugates in solution. We have used this method to examine the kinetics of sulphation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The efficiency of recovery of the sulphate conjugates was greater than 86%. The method is applicable to the quantitative study of sulphate conjugation of any substrate which forms a sulphate conjugate that is soluble in the presence of barium, without the need for standards or radiolabelled sulphate acceptors.

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

The roles of glucagon, insulin and glucocorticoid hormones in the effects of sublethal doses of endotoxin on glucose homeostasis in rats.

The effects of sub-lethal doses of endotoxin on plasma glucose, glucagon, insulin, glucocorticoids and non-esterified fatty acids (NEFA) were determined in rats. Endotoxin did not change the plasma concentration of glucocorticoids, but blocked the effects of elevated glucocorticoid hormone concentrations on both plasma glucose and hepatic tryptophan dioxygenase activity. Endotoxin increased the plasma concentrations of glucose, glucagon and insulin in rats with basal glucocorticoid concentrations, and changed the observed relationships between glucose, glucagon and insulin in a manner consistent with an increased sensitivity of glucagon secretion to lowered glucose concentrations. At the highest dose of endotoxin used, 20 mg/kg over 6 hr, a substantial decrease (greater than 7-fold) in the insulin/glucagon ratio provides evidence for changes in basal (as opposed to hormone-stimulated) glucose production and/or utilisation in vivo.

Uptake of acetaminophen (paracetamol) by isolated rat liver cells.

The characteristics of the uptake of acetaminophen (N-acetyl-p-aminophenol or paracetamol, APAP) in incubations of isolated rat liver cells were consistent with diffusion of the drug being the predominant mechanism of APAP influx in these cells at concentrations above 0.5 mM. At lower substrate concentrations (below 0.5 mM) a saturable component was apparent. Both uptake processes could have a role in the control of the metabolism of APAP, because, at low concentrations, there was no intracellular accumulation of unconjugated drug, all the APAP entering the cell being converted to sulphate and glucuronide. After addition of drug, there was a lag phase of approximately 5 min before APAP-glucuronide and APAP-sulphate appeared in the incubation medium; during this time both conjugates accumulated inside the cells. These results have implications for our understanding of the mechanisms of APAP transport, and indicate how these processes may affect the drug's overall metabolism.

Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis.

Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-alpha (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6 x DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was covered rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrate, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71 +/- 0.04 l/kg and the clearance was 0.32 +/- 0.02 l/h-1/kg-1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.

A microtiter-plate assay of nitric oxide synthase activity.

We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing the spectral shift in optical absorbence between the wavelengths 405 and 420 nm on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). This is a high-throughput assay permitting 96 or 384 simultaneous kinetic measurements and is ideal for the study of NOS inhibitors and their time dependence. It is also possible to measure enzyme rates under different conditions simultaneously for the study of the cofactor and substrate dependence of NOS preparations. The assay requires approximately 10 pmol/min of NOS activity to achieve a-l moD/min rate.

Control of phenylalanine and tyrosine metabolism by phosphorylation mechanisms.

A system for the parallel determination of enzyme phosphorylation and expressed activity in rat liver cells, and its application to studies of phenylalanine hydroxylase and tyrosine aminotransferase, is described. Phenylalanine hydroxylase is phosphorylated by agents which stimulate cyclic AMP- and Ca2+-dependent protein kinase activity. The phosphorylation site(s) appear to be the same for both kinases. Phosphorylation is accompanied by increased metabolic flux at low, physiologically relevant, substrate concentrations. Insulin and spermine both inhibit the phosphorylation of the enzyme, possibly by increasing dephosphorylation. Tyrosine aminotransferase is phosphorylated in liver cell incubations but the rate is slow and insensitive to additions to the medium. No parallel changes in flux could be detected. Both enzymes are subject to complex regulatory mechanisms, short- and long-term. Their activities may be coordinated in vivo by control exerted at the level of the plasma membrane where both amino acids share the same transport processes. Determination of the control coefficients for the several components indicates that membrane transport may be a major limitation on flux.

Inducible nitric oxide synthase is involved in the mechanisms of cocaine enhanced neuronal apoptosis induced by HIV-1 gp120 in the neocortex of rat.

Cocaine, often abused by human immunodeficiency virus (HIV) infected patients, has been suggested to worsen the HIV associated dementia via unknown mechanisms. Here we report that subchronic treatment with a dose of cocaine (30 mg/kg i.p.), unable per se to cause neuronal death, increases the number of apoptotic cells typically observed in the neocortex of rats treated with HIV-1 gp120 (100 ng given i.c.v.). A pre-treatment with MK801 (0.3 mg/kg i.p.), a NMDA receptor antagonist, L-NAME (10 mg/kg i.p.) and 7-nitroindazole (50 mg/kg i.p.), two specific inhibitors of NOS, or with 1400 W (1 mg/kg s.c.), a selective inhibitor of inducible NOS (iNOS), minimized neurotoxicity by combined administration of cocaine and gp120 thus implicating iNOS. This conclusion is supported by the evidence that cocaine increases brain neocortical citrulline, the co-product of NO synthesis.

Factors affecting the DNA damaging activity of superoxide and nitric oxide.

Nitric oxide and superoxide are formed endogenously and can react with each other and with other molecules to form a range of secondary and tertiary products. Some of these (e.g., peroxynitrite) are potent DNA-damaging agents and others (e.g., S-nitrosoglutathione) can act as reservoirs of the reactive species. Although the chemistry of these processes is now becoming understood, the question of which products are significant in vivo is not necessarily clear. To investigate these processes we have developed a cell-free version of the Comet assay, where the DNA from isolated nuclei is treated in agar on a microscope slide, following lysis. This offers an exceptionally sensitive assay for strand breakage in free DNA. Despite being present as a scavenger in the cell at millimolar levels, glutathione can act as a DNA-damaging pro-oxidant. Under appropriate conditions, glutathione-mediated damage is suppressed by superoxide dismutase and we suggest that superoxide may be a direct damaging agent, whose activity can be masked because of the involvement of superoxide in indirect mediation of damage or because of concomitant presence of hydroxyl radical.