Expression of nitric oxide synthase 2 and cyclooxygenase-2 in swine experimentally infected with Actinobacillus pleuropneumoniae

The expression of inflammatory mediators was examined in pigs experimentally infected with Actinobacillus pleuropneumoniae. The activity of nitric oxide synthase 2 (NOS2) and cyclooxygenase-2 (COX-2) was determined by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) in bronchoalveolar lavage fluid in response to A. pleuropneumoniae in vivo. By in situ hybridization and immunohistochemistry, both NOS2 and COX-2 enzymes were detected in neutrophils and macrophages that had infiltrated into alveolar spaces. The sharp increase in PGE2 concentration preceded the increase in the concentrations of NO. NO levels were highly correlated with PGE2 level (rs=0.7218, P <0.05). The NO levels were positively correlated with lung lesion scores (rs=0.9087, P <0.05) until 24 hours postinoculation (hpi) as were the lung lesion scores and PGE2 levels (rs=0.925, P <0.01). High levels of PGE2 produced by COX-2 are generated in early infection (6 hpi). However, in later stages of infection (12-36 hpi), there is participation of NO and PGE2 accompanied by coinduction of both NOS2 and COX-2.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

Effects of nitric oxide donor and nitric oxide synthase inhibitor on ruminal contractions in conscious sheep

The present study was planned to evaluate a role of nitric oxide (NO) in the regulation of regular ruminal contractions in conscious sheep. Intravenous infusion of S-nitroso-acetyl-DL-penicillamine (SNAP) at doses of 3-30 nmol kg(-1) min(-1)for 30 minutes inhibited both the amplitude and frequency of ruminal contractions in a dose-dependent manner. However, intravenous infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) at doses of 0.3-3.0 micromol kg(-1) min(-1)did not alter the basal tone of intraruminal pressure and the amplitude of ruminal contractions. The frequency of contractions was slightly inhibited by L-NAME infusion at 1.0 micromol kg(-1)min(-1). The effects of L-NAME were abolished by simultaneous infusion of L -arginine at 30 micromol kg(-1) min(-1). These results suggest that exogenous NO can diminish the ruminal contractions, while endogenous NO is not involved in the regulatory mechanism of basal tone and regular phasic contractions of the rumen in healthy sheep.     

The equine alveolar macrophage: Functional and phenotypic comparisons with peritoneal macrophages

Alveolar macrophages (AMs) constitute the first line of defence in the lung of all species, playing a crucial role in the regulation of immune responses to inhaled pathogens. A detailed understanding of the function and phenotype of AMs is a necessary pre-requisite to both elucidating their role in preventing opportunistic bacterial colonisation of the lower respiratory tract and developing appropriate preventative strategies. The purpose of the study was to characterise this important innate immune cell at the tissue level by making functional and phenotypic comparisons with peritoneal macrophages (PMs). We hypothesised that the tissue of origin determines a unique phenotype of AMs, which may constitute an appropriate therapeutic target for certain equine respiratory diseases. Macrophages isolated from the lung and the peritoneal cavity of 9 horses were stimulated with various toll like receptor (TLR) ligands and the production of nitrite, tumour necrosis factor alpha (TNFα), interleukin (IL) 10 and indoleamine 2,3-dioxygenase (IDO) were measured by the Griess reaction and enzyme linked immunosorbent assay (ELISA) and/or quantitative polymerase chain reaction, respectively. Cells were also compared on the basis of phagocytic-capacity and the expression of several cell surface markers. AMs, but not PMs, demonstrated increased TNFα release following stimulation with LPS, polyinosinic polycytidylic acid (Poly IC) and heat-killed Salmonella typhinurium and increased TNFα and IDO mRNA expression when stimulated with LPS. AMs showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and TLR4, whereas PMs showed high expression of TLR4 only. AMs, but not PMs, demonstrated efficient phagocytic activity. Our results demonstrate that AMs are more active than PMs when stimulated with various pro-inflammatory ligands, thus supporting the importance of the local microenvironment in the activation status of the macrophage. This information provides a valuable knowledge base on which to improve our understanding of the role of macrophages and their microenvironment in equine innate immunity.     

Effect of strenuous exercise stress on chemiluminescence response of equine alveolar macrophages

Bronchoalveolar lavage samples were collected using a fibreoptic endoscope from horses at specified times before and after single bouts of exercise. Lucigenin-dependent phagocytic chemiluminescence was used to assess the effect of exercise on the alveolar macrophage metabolic activity in response to stimulation by opsonised zymosan. A profound suppressive effect on the chemiluminescence production was present throughout the first three days after exercise. However, the cellular composition of lavage fluids was not altered by the exercise. It is suggested that strenuous exercise may jeopardize the antimicrobial function of alveolar macrophages which may lead to an increase in susceptibility to infection.     

一氧化氮合酶阳性神经元在大鼠体内的分布与一氧化氮的功能

一氧化氮 (ＮＯ)是一种最新发现的、哺乳动物中最小、最轻并具有独特理化性质和生物学活性的信息和效应分子 ,能激活靶细胞中的鸟苷酸环化酶 (ＧＣ) ,提高环一磷酸鸟苷(ｃＧＭＰ)浓度 ,发挥一系列生物学作用 ,现已成为研究热点之一。ＮＯ广泛存在于神经系统、心血管系统、免疫系统、消化系统、生殖系统与呼吸系统等的细胞内 ,是传递神经信息、调节血压以及机构防御等一系列生命活动必不可少的生物信使。因此 ,内源性ＮＯ的生物学研究、ＮＯ在动物模型大鼠体内的分布及其功能的确定 ,将有助于在动物医学和人类临床医学领域进一步阐明机体某些…     

Balb/c小鼠肺组织一氧化氮及一氧化氮合酶的动态变化

为探讨Balb/c小鼠正常生理状况下肺组织中一氧化氮自由基含量以及一氧化氮合酶活性的动态变化,采用电子自旋共振法直接测定了一氧化氮自由基含量,采用分光光度计法测定了一氧化氮合酶的活性.结果表明:在第3,6,7,12天Balb/c小鼠肺组织的一氧化氮自由基含量、一氧化氮合酶活性均无显著性差异(P＞0.05).说明生理状态下,Balb/c小鼠肺组织一氧化氮自由基的产生维持动态平衡.     

PAF increases phagocytic capacity and superoxide anion production in equine alveolar macrophages and blood neutrophils

Phagocytosis exerted by alveolar macrophages and neutrophils is crucial in the clearance of exogenous particles deposited in the airways. Therefore, substances that activate these phagocytes in the airways can exert important effects on the particle clearance rate. PAF, particularly, was proved to be a potent activator of several immune cells and was shown to be present in the equine lower airways in specific conditions, such as after exercise. The present study aimed to investigate if PAF is able to increase the phagocytic capacity and the production of superoxide anion in equine alveolar macrophage and blood neutrophils. The results show that PAF increased these parameters in both phagocytes even in concentrations as low as 0.1 and 1.0 nM. On that ground, the present work suggests that PAF is involved in the process of particle clearance in equine lower airways.     

Evidence of nitric oxide synthase 2 activity in swine naturally infected with Actinobacillus pleuropneumoniae

Evidence of nitric oxide synthase (NOS) 2 activity was determined by formation of nitrotyrosine (a reaction product of peroxynitrite) and by activation of poly(ADP-ribose) synthetase (PARS) in NOS2-expressed pleuropneumonic lungs from 20 pigs naturally infected with Actinobacillus pleuropneumoniae using immunohistochemistry. Intense immunostaining for nitrotyrosine residue was seen within the lung lesions from A. pleuropneumoniae-infected pigs, but it was minimal in the unaffected parts of the lung from A. pleuropneumoniae-infected pigs and in the normal lung from control pigs. Staining was especially strong in neutrophils and macrophages in the periphery of the lesions and within the alveolar spaces. There was close cell-to-cell correlation when serial sections were examined by immunohistochemistry for NOS2 and nitrotyrosine in each of the 20 lung samples. Expression of PARS was always present within inflammatory lesions but was minimal in the unaffected lung of A. pleuropneumoniae-infected pigs. Macrophages in alveolar spaces frequently exhibited strong staining for PARS. Colocalization of nitrotyrosine and PARS antigen was especially prominent in macrophages in the periphery of lesions. NOS2 expression in pleuropneumonic areas associated with protein nitrosation and PARS suggests that NOS2 is functionally active during infections caused by A. pleuropneumoniae.     

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

日粮铜对肉鸡肾脏一氧化氮产生及一氧化氮合酶活性的影响

铜是动物体内一种必需的微量元素,在多种代谢途径中有重要的生物学作用.早期的研究结果表明,提高饲料中铜的含量,可以促进动物生长.但是过量添加铜也可以损害肝脏、肾脏等器官[1-2].一氧化氮(NO)作为一种强有力的内皮细胞舒血管因子,其功能已越来越受到人们的重视.NO对肾脏具有保护和损伤的双重作用.一方面具有扩张动脉,抑制血小板粘附、聚集,抗血栓形成,疏通微循环,增加供血,保护细胞的作用.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Melatonin regulates nitric oxide synthase expression in ischemic brain injury

Nitric oxide (NO) is produced by three NO synthases (NOS), iNOS, eNOS, and nNOS. Production of NO by iNOS plays key roles in neurodegeneration, while eNOS is a protective enzyme. This study investigated the neuroprotective effect of melatonin and the levels of NOS isoforms induced by melatonin in ischemic brain injury. Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brain samples were collected at 24 hr after the onset of occlusion. Results confirmed that melatonin significantly reduces infarct area. Western blot analysis was used to evaluate the expression levels of iNOS, eNOS, and nNOS. The level of iNOS and nNOS increased in vehicle-treated animals, while melatonin prevented injury-induced increase of iNOS. In contrast to iNOS levels, eNOS levels decreased in vehicle-treated animals, while melatonin prevented the injury-induced decrease of eNOS. This study provides further evidence that melatonin exerts neuroprotective effects, and the regulation of NOS isoforms by melatonin may contribute to the neuroprotective effects.