ovalbumin and pimagedine

One mechanism by which fructose could exert deleterious effects is through intestinal formation and absorption of pro-inflammatory advanced glycation endproducts via the Maillard reaction. We employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that advanced glycation endproducts (AGEs) are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions.. OVA was subjected to simulated gastric and intestinal digestion using standard models, in presence of fructose or glucose (0-100mM). Peptide fractions were analyzed by fluorescence spectroscopy and intensity at Excitation: λ370nm, Emission: λ 440nm.. AGE adducts formed between fructose and OVA, evidenced by the peptide fractions (<5kDa) at times (30min) and concentration ranges (10mM) plausibly found in the intestines, whereas no reaction occurs with glucose. The reaction was inhibited by chlorogenic acid at concentrations compatible with those found in the gut. The reaction was also inhibited by aminoguanidine, a specific antiglycation agent.. Our study showed fructose-AGE formation on a ubiquitous dietary protein under model physiological conditions. Our study also suggests ways to decrease the damage: enteral fructose-AGE formation may be partially inhibited by co-intake of beverages, fruits and vegetables with concentrations of phenolics high enough to serve as anti-glycation agents.

Topics: Chlorogenic Acid; Digestion; Duodenum; Fructose; Gastric Juice; Glucose; Glycation End Products, Advanced; Guanidines; Maillard Reaction; Ovalbumin

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Topics: AMP-Activated Protein Kinases; Animals; Asthma; Blotting, Western; Bronchoalveolar Lavage Fluid; Diet, High-Fat; Enzyme Inhibitors; Eosinophils; Guanidines; Hypoglycemic Agents; Inflammation; Insulin Resistance; Lung; Male; Metformin; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type II; Obesity; Ovalbumin; Promoter Regions, Genetic; Protein Binding; Transcription Factor RelA; Tumor Necrosis Factor-alpha

The competition between arginases and NO synthases (NOS) for their common substrate L-arginine can be important in the airways hyperreactivity. We investigated the effect of the simultaneous modulation of arginase and NOS activities in allergen-induced airways hyperreactivity. We analysed the response of tracheal and lung tissue smooth muscle to histamine or acetylcholine after administration N(ω)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and N(ω)-hydroxy-L-arginine (NOHA) in the combinations in in vitro conditions. The results show the decrease of ovalbumin-induced hyperreactivity after inhibition of arginase activity with NOHA. A supplementation of L-arginine caused favourable effect on the airway smooth muscle response. We found the airway reactivity decrease on the whole if we used the combination of NOS and arginase inhibitors. The inhibition of both types of enzymes caused more expressive effect in tracheal smooth muscles. We recorded the difference in the response to histamine or acetylcholine. The simultaneous inhibition of iNOS (with AG) and arginase (with NOHA) evoked the most expressive effect. Results show the importance of competition of both types enzymes - NOS and arginase for the balance of theirs activities in the control of airways bronchomotoric tone in the conditions of the airways hyperreactivity.

Topics: Acetylcholine; Allergens; Animals; Arginase; Arginine; Bronchial Hyperreactivity; Enzyme Inhibitors; Guanidines; Guinea Pigs; Histamine; Lung; Male; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Ovalbumin; Substrate Specificity; Trachea

Herewith we investigated the role of nitric oxide synthase (NOS)-II in the establishment of oral tolerance induced by low antigen dose. To accomplish this, we used a rat model of oral tolerance induced by intragastric administration of low doses of ovalbumin (OVA). NOS-II was inhibited in vivo during the onset of tolerance by intraperitoneal (i.p.) treatment with aminoguanidine (AMG), a selective NOS-II inhibitor. Four experimental groups were generated: (TOL), tolerised rats, receiving OVA but no AMG; (TAG), rats tolerised with OVA and simultaneously receiving AMG i.p.; (CAG), controls treated with AMG but no oral antigen; and (CONT), controls receiving neither OVA nor AMG treatment. The state of oral tolerance was evaluated in all groups by analysing several immune parameters upon subcutaneous administration of OVA in Freund's complete adjuvant. First, we were able to determine that NOS-II inhibition altered the TH1/TH2 balance in tolerised rats, driving the TH2 anti-OVA response in TOL rats towards TH1 in TAG animals, which showed enhanced delayed hypersensitivity responses. Second, splenocyte cultures from TAG rats showed lower levels of IL-10 production compared to TOL samples as determined by ELISA analysis. Last, we detected the presence of a functional distinct Tr1 regulatory T cell population in spleen samples recovered from TAG animals. Contrary to what happened with TOL Tr1 cells, the levels of Tr1 cells in TAG samples were modified by in vitro stimulation with OVA. All together, these data indicate a preponderant role for NOS-II in the process of oral tolerance induced by low antigen dose.

Topics: Administration, Oral; Animals; Cells, Cultured; Chickens; Enzyme Inhibitors; Female; Guanidines; Immune Tolerance; Injections, Intraperitoneal; Models, Animal; Nitric Oxide Synthase Type II; Ovalbumin; Rats; Rats, Wistar; Th1 Cells; Th2 Cells

The decreased L-arginine bioavailability, the basic substrate for nitric oxide synthesis, can be one of the factors contributing to the airways hyperreactivity.. We investigated the effect of various inhibitors of the enzyme activities utilizing L-arginine in a guinea pig model of experimental ovalbumin-induced airway hyperreactivity.. We used the in vivo pre-treatment with non-specific inhibitor of NO synthase No-nitro-L-arginine metylester (L-NAME) and relatively specific inhibitor of inducible NO synthase--aminoguanidine. Inhibitors were administered in one-shot (on the 14th day, 30 minutes before the inhalation of ovalbumin) or in a long-time regime (during the whole period of sensibilization by ovalbumin--14 days). We administered the inhibitor of arginase Nomega-hydroxy-L-arginine (NOHA) to the tracheal and lung tissue smooth muscle strips from sensibilized animals.. We observed an increase in the tracheal smooth muscle response to histamine in animals that received an inhalation dose of L-NAME (40 mg/kg b.w.) or aminoguanidine (50 mg/kg b.w.) 30 minutes before the inhalation of ovalbumin but did not evoke any significant difference in the reactivity of lung tissue smooth muscle. Tracheal smooth muscle responded with enhanced contraction amplitude to histamine after chronic pre-treatment with L-NAME or aminoguanidine. The inhibition of arginase with NOHA in vitro decreased the tracheal and lung tissue smooth muscle reactivity to histamine.. The results suggest that NO-synthase isoforms as well as arginase are involved in the production of NO and in the control of bronchomotoric tonus (Fig. 4, Tab. 2, Ref. 31). Full Text (Free, PDF) www.bmj.sk.

Topics: Allergens; Animals; Arginase; Arginine; Bronchial Hyperreactivity; Enzyme Inhibitors; Guanidines; Guinea Pigs; In Vitro Techniques; Lung; Male; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type I; Ovalbumin; Trachea

A correlation between stressful events experienced by the mother during pregnancy and progression of respiratory disease in descendants has been reported. Prenatal exposure to lipopolyssacharide (LPS) reduces allergic airway inflammation in mice offspring. In this study we investigated whether reduction of airways inflammation by maternal LPS exposure involves activation of inducible nitric oxide synthase (iNOS) at prenatal life. Since LPS also induces the release of TNF-alpha, and that this cytokine has been implicated in pathogenesis of asthma, we also evaluated whether TNF-alpha plays a role in the allergic airways inflammation by maternal LPS exposure. Pregnant rats were pretreated with the iNOS inhibitor aminoguanidine (50mg/rat per day; given from day 13 of gestation up to delivery) before exposure to LPS (7mug/kg, given at day 17 of gestation). At adult ages, female and male offspring were sensitized with ovalbumin (OVA), and 14 days later they were challenged with this allergen. OVA challenge in sensitized offspring increased markedly the eosinophil number in bronchoalveolar lavage (BAL) fluid at 48h compared with the non-sensitized group. However, the eosinophil number was largely reduced in offspring from maternal LPS exposure, irrespective of offspring gender. Maternal pretreatment with aminoguanidine fully reversed the eosinophil suppression by LPS. The maternal LPS exposure also reduced the eosinophil number in bone marrow and peripheral blood of offspring, but this was not affected by aminoguanidine. No differences in TNF-alpha levels in BAL fluid were found. In conclusion, our study shows that maternal LPS exposure markedly reduces allergic airways eosinophil recruitment in adult offspring by mechanisms possibly involving iNOS activation.

Topics: Animals; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Enzyme Activation; Eosinophils; Female; Guanidines; Leukocyte Count; Lipopolysaccharides; Male; Maternal Exposure; Maternal-Fetal Exchange; Nitric Oxide Synthase Type II; Ovalbumin; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Eosinophilia; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.

Topics: Amidines; Animals; Arginase; Asthma; Bronchoalveolar Lavage Fluid; Down-Regulation; Drug Synergism; Enzyme Inhibitors; Guanidines; Heterocyclic Compounds, 2-Ring; Lipid Peroxides; Lung; Male; Nitric Oxide Synthase Type II; Ovalbumin; Rats; Rats, Inbred BN; Superoxide Dismutase

In the present study we investigated the lymphocytes infiltration and other parameters of allergic lung inflammation comparing mice submitted to acute suppression of nitric oxide synthesis with mice deficient in inducible nitric oxide synthase (NOS2-/-) gene. At weekly intervals C57Bl/6 mice, wild type and NOS2-/- were sensitized twice with ovalbumin-alumen and challenged twice with ovalbumin aerosol and lungs examined 24 h later. In wild type mice, treatment with nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine-methyl-ester (L-NAME) or aminoguanidine (i.p., 30 min before each ovalbumin challenge) caused a significant decrease in bronchoalveolar lavage cell number: eosinophils (90%), lymphocytes NK1.1+ (70%), Tgammadelta+ (50%), CD4+ (55%), CD8+ (60%) and B220+ (65%). Both inhibitors abolished airway hyperreactivity and significantly reduced mucus secretion (L-NAME 64%; aminoguanidine 58%). Surprisingly, in NOS2-/- mice these parameters of allergic lung inflammation were not significantly different when compared with wild type mice. In addition, treatment of NOS2-/- mice with L-NAME or aminoguanidine did not affect these parameters. Thus, acute inhibition of NOS2 activity inhibits asthma-like responses but absence of NOS2 has no affect.

Topics: Animals; Antigens; Antigens, Ly; Antigens, Surface; Asthma; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; CD4 Antigens; CD8 Antigens; Enzyme Inhibitors; Eosinophils; Flow Cytometry; Guanidines; Lectins, C-Type; Leukocyte Common Antigens; Leukocyte Count; Lung; Lymphocyte Count; Lymphocytes; Methacholine Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; NK Cell Lectin-Like Receptor Subfamily B; Ovalbumin; Proteins

Here, we studied the effect of aminoguanidine (AG) treatment, a nitric oxide synthase (NOS)-2 inhibitor, during the immune response against intranasal administration of ovalbumin (OVA) mixed with cholera toxin (CT) in BALB/c mice. NOS-2 mRNA was detected by reverse transcription-PCR (RT-PCR) in samples of lungs and turbinates early post-inoculation of the antigen. Animals intranasally treated with AG, showed an increase in the levels of seric specific IgG and IgM. A higher IgG1/IgG2a ratio against OVA was also observed in sera of same animals. Moreover, high levels of specific IgA were detected in samples of pulmonar washings obtained from treated animals. On the contrary, treated animals showed a lower DTH response while splenocytes obtained from the same animals showed a reduced proliferative capability against OVA compared to controls. Finally, RT-PCR analysis showed increased expression of TGF-beta in turbinates, lungs and cells from pulmonar washings obtained from AG treated mice. Taken together, these data suggest a role of nitric oxide (NO) in modulating the primary immune response against intranasal antigens.

Topics: Administration, Intranasal; Animals; Cholera Toxin; Guanidines; Immunity, Cellular; Immunity, Mucosal; Lung; Mice; Nitric Oxide; Nitric Oxide Synthase; Ovalbumin; Transforming Growth Factor beta; Turbinates

Using a guinea pig model of acute allergic asthma, we recently established that a deficiency of nitric oxide (NO) contributes to airway hyperreactivity (AHR) after the early asthmatic reaction (EAR) and that restoration of NO activity may contribute to the (partial) reversal of AHR after the late asthmatic reaction (LAR). In the present study, we investigated the role of iNOS-derived NO in the regulation of AHR to histamine after the LAR. Inhalation of a selective dose of the specific iNOS inhibitor aminoguanidine (0.1 mM, 3 min) had no effect on basal airway reactivity to histamine in unchallenged, ovalbumin-sensitized animals and did not affect the allergen-induced AHR after the EAR. By contrast, this dose of aminoguanidine significantly potentiated the partially reduced AHR after the LAR to the level of AHR observed after the EAR, indicating that induction of iNOS during the LAR contributes to the reversal of AHR. Inhalation of a higher aminoguanidine concentration (2.5 mM) shortly before the onset of the LAR diminished the AHR after the LAR and reduced the number of neutrophils, lymphocytes, and ciliated epithelial cells in the bronchoalveolar lavage at this time point. The results indicate that iNOS-derived NO may have both beneficial and detrimental effects on allergen-induced AHR to histamine after the LAR by functional antagonism of histamine-induced bronchoconstriction, and by promoting airway inflammation and epithelial damage on the other hand, respectively.

Topics: Acute Disease; Adjuvants, Immunologic; Allergens; Animals; Asthma; Bronchial Hyperreactivity; Bronchitis; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Female; Guanidines; Guinea Pigs; Histamine; Leukocyte Count; Lymphocyte Count; Male; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Ovalbumin

The role of nitric oxide in allergic conjunctivitis was studied in a guinea pig model. The eyes of sensitized guinea pigs were challenged with ovalbumin (20 micrograms per eye) or histamine (20 micrograms per eye). Synthesis of nitric oxide (NO) was inhibited using L-NAME (200 micrograms per eye) or aminoguanidine (200 micrograms per eye). The formation of conjunctival edema was graded and levels of nitrite, a breakdown product of nitric oxide were measured in lavage fluid. Conjunctival vasopermeability was determined by measuring the albumin concentration in the fluid on the surface of the eye (lavage fluid). Animals were treated with sodium nitroprusside (SNP) or phenylephrine after which histamine induced conjunctival vasopermeability changes were measured. Drugs were administered topically with the other eye serving as a control. Both ovalbumin and histamine produced a marked inflammatory response including hyperaemia and edema. At the top of the inflammatory response occurring 30 min after challenge, increased levels of nitrite, a breakdown product of NO, were measured in lavage fluid. Prophylactic treatment with L-NAME or aminoguanidine resulted in a significant inhibition of the NO synthesis. Both L-NAME and aminoguanidine decreased conjunctival vascular permeability and edema formation significantly. Administration of SNP resulted in a marked dilatation of conjunctival blood vessels and produced a dose-dependent increase of vascular permeability. Addition of SNP to histamine significantly enhanced conjunctival edema and potentiated vascular permeability. These results indicate that NO is produced in the acute phase of allergic conjunctivitis and mediates vasodilatation after topical provocation with ovalbumin or histamine in sensitized guinea pigs. The resulting increase of the conjunctival blood flow subsequently increases the vascular permeability and enhances conjunctival edema formation. Inhibition of NO synthesis leads to a reduction of conjunctival hyperaemia and subsequently reduces the formation of edema.

Topics: Animals; Arginine; Capillary Permeability; Conjunctival Diseases; Conjunctivitis, Allergic; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Female; Guanidines; Guinea Pigs; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Ovalbumin; Phenylephrine

The aim of the study was to investigate whether or not the epithelium plays an active role in histamine metabolism, via the histaminase and/or methyltransferase pathways. Isolated tracheas from guinea pigs sensitized to egg albumin (EA) were used. The epithelium was either left intact or removed from the preparations. The tracheal tubes were mounted in a chamber, allowing estimation of smooth muscle tension, and perfused with buffer. In some experiments the perfusate was collected for determination of histamine and methylhistamine. Mepyramine was used to evaluate the contribution of histamine to the EA-induced contraction. Mepyramine reduced the contraction by 90% when the epithelium was removed; with intact epithelium the reduction was 47%. Aminoguanidine, a histaminase inhibitor, significantly potentiated the response to EA when the epithelium was left intact. Traces of methylhistamine were detected in tissue extracts and perfusates. We conclude that histaminase is present in the preparation and that it can contribute to the inhibitory effect of the epithelium by means of histamine degradation.

Topics: Amine Oxidase (Copper-Containing); Animals; Antigens; Epithelium; Female; Guanidines; Guinea Pigs; Histamine; Methylhistamines; Muscle Contraction; Ovalbumin; Pyrilamine; Trachea

The difference in biological effects and distribution when aminoguanidine sulfate (AGS) was injected into different sites of developing eggs was studied. (I) Five mg to 20 mg of AGS were injected once into the air sac (AS), albumen (AL) or yolk sac (YS) from the 4th to the 7th day of incubation. Observation continued up to the 18th day of incubation. In the AS group, total mortality was the highest and external abnormalities were most frequent; however, abnormal findings in liver were infrequent and slight. In the YS and particularly in the AL group, on the other hand, liver abnormalities such as low weight, degeneration, necrosis, increase of mitosis, delay of development and fibrosis were found. These are considered as specific effects of AGS. These frequencies were high when AGS was injected on the 4th and 5th day of incubation. (II) Twenty mg injection of AGS into the AS, AL, or YS on the 5th or 8th day of incubation was made for a distribution study. AGS concentration was determined in the various compartments as various times after injection. Distribution of AGS in the AS group was significantly different from that in the AL and YS groups. The AL and YS groups, however, differed a little. The exact route which AGS reaches from the site of injection to the chick embryos could not be determined, but it is concluded that the albumen is the best site for the injection of AGS to investigate its specific effects on liver.

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Guanidines; Injections; Liver; Ovalbumin; Time Factors; Yolk Sac