Higher Nitric Oxide Production Research Articles

Effects of vitamin D-induced supernatant of placental explants from preeclamptic women on oxidative stress and nitric oxide bioavailability in human umbilical vein endothelial cells.

The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H2O2) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO3 -), and nitrite (NO2 -) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO3 -, NO2 -, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells.

Alleviated Immunological Activities of Wistar Rat Peritoneal Neutrophils and Macrophages by Polysaccharide Rich Extract from the Fruit Bodies of Microporus vernicipes (Polyporales)

Aim: This study aimed to assess the effect of polysaccharides from fruit body of Microporous vernicipes (Polyporales) on immunological activities of macrophages and neutrophils.
 Methodology: Polysaccharides were extracted using hot water (PHW) and cool water (PCW) and tested on the activities of peritoneal macrophage and neutrophil in response of lipopolysaccharides. The effect of the polysaccharides was examined In vitro to assess the phagocytic activities by evaluating the vacuole formation, nitroblue tetrazolium dye reduction, lysosomal enzyme activity, nitric oxide, myeloperoxidase in neutrophil and on neutrophil adherence assay.
 Results: The cool and hot water polysaccharides of Microporus vernicipes significantly improved the activities of macrophages and neutrophils as demonstrated by an elevated the formation of vacuole following by a high production of nitric oxide, lysosomal enzyme and superoxide. Those polysaccharides extracts of Microporous vernicipes also caused an increase in release of myeloperoxidase by neutrophils. Results also showed that neutrophils treated with polysaccharides extracts of Microporous vernicipes adhered more on a plastic surface than those untreated. It has been also found that polysaccharides of Microporous vernicipes may have cytotoxic effect at high concentration with IC50 of 2.28 and 8.15 mg/mL for PHW and PCW respectively.
 Conclusion: The result of this study indicates the immunostimulatory activity of polysaccharides of Microporus vernicipes by stimulating activities of macrophages and neutrophils.

Endogenous flux of nitric oxide: Citrulline is preferred to Arginine.

Both arginine (Arg) and its precursor citrulline (Cit) have received much interest in the past two decades because of their potential effects on whole-body nitric oxide (NO) production and augmentation of NO-dependent signalling pathways. However, the usefulness of Arg supplementation for NO production is questionable because of its high splanchnic first pass metabolism (FPM), which limits its systemic availability. Both hepatic- and extrahepatic arginases critically limit the availability of Arg for the NO synthase enzymes (NOSs) and therefore, a limited amount of oral Arg can reach the systemic circulation for NO synthesis. Arg also has some undesired effects including induction of arginase activity, an increase of urea levels, a decrease of cellular uptake of Cit and decrease of recycling of Arg from Cit. In contrast, Cit has more availability as an NO precursor because of its high intestinal absorption, low FPM and high renal reabsorption. At the cellular level, co-localization of Cit transport systems and the enzymes involved in the Cit-Arg-NO pathway facilitates channelling of Cit into NO. Furthermore, cells preferably use Cit rather than either intra- or extracellular Arg to improve NO output, especially in high-demand situations. In conclusion, available evidence strongly supports the concept that Cit leads to higher NO production and suggests that Cit may have a better therapeutic effect than Arg for NO-disrupted conditions.

Neopterin derivatives - a novel therapeutic target rather than biomarker for atherosclerosis and related diseases.

This review provides an updated overview of the emerging roles of neopterin derivatives in atherosclerosis. Neopterin, a metabolite of guanosine triphosphate, is produced by interferon-γ-activated macrophages and is expressed at high levels in atheromatous plaques within the human carotid and coronary arteries as well as in the aorta. Plasma concentrations of neopterin are higher in patients with carotid, cerebral, and coronary artery diseases as well as aortic aneurysm. The concentration of neopterin is positively correlated with the severity of coronary artery disease. However, a prospective cohort study showed that neopterin contributes to protection against plaque formation in carotid arteries in patients with atherosclerosis. Moreover, using both in vitro and in vivo experiments, a recent study has shown the atheroprotective effects of neopterin. Neopterin suppresses the expression of monocyte chemotactic protein-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in endothelial cells, and thereby suppresses the adhesion of monocytes to endothelial cells. It also suppresses the inflammatory phenotype of monocyte-derived macrophages. In addition, neopterin suppresses oxidized low-density lipoprotein-induced foam cell formation in macrophages and the migration and proliferation of vascular smooth muscle cells. Neopterin injection into apolipoprotein E-deficient (Apoe-/-) mice suppresses the development of atherosclerotic lesions. A neopterin derivative tetrahydroneopterin (BH4), also known as a cofactor for nitric oxide (NO) synthases, suppresses atherosclerosis and vascular injury-induced neointimal hyperplasia in Apoe-/- mice. BH4 administration improves endothelial dysfunction in patients with coronary artery disease. These findings suggest that neopterin production may increase to counteract the progression of atherosclerosis, as neopterin contributes to atheroprotection. Otherwise, the increased neopterin levels in atherosclerosis may reflect a compensatory mechanism associated with inducible NO synthase upregulation in macrophages to supply BH4 for high output NO production caused by decreased endothelial NO synthase in atherosclerosis. Therefore, neopterin derivatives are a novel therapeutic target for atherosclerosis and related diseases.

In vitro model using cytokine cocktail to evaluate apoptosis in Min6 pancreatic beta cells

IntroductionDevelopment of therapy options for treatment of type 1 diabetes mellitus is hampered by non-availability of appropriate experimental models that can exactly mimic the in vivo situation. Apoptosis of beta cells by T cells and cytokine action leads to loss of beta cells. We propose a simple and elegant model using cytokine cocktail of TNF-α, IFN-γ and IL-1β, the major cytokines responsible for apoptosis in Min6 beta cell line. MethodsA cocktail of TNF-α, IFN-γ and IL-1β was used to induce apoptosis in Min6 beta cell line. Apoptosis was assessed by flow cytometry using CytoFLEX (Beckman Coulter). The destruction of beta cells is through production of nitric oxide (NO), oxidative stress and change in mitochondrial membrane permeability. NO was measured using Griess reagent. Oxidative stress was assessed using 2',7'-dichlorofluorescein diacetate, a cell-permeable fluorogenic dye and mitochondrial membrane potential was determined on the basis of retention of rhodamine 123 using flow cytometer. Results and discussionVery low concentration of the cocktail viz. TNF-α 25 ng/ml, IFN-γ 25 ng/ml and IL-1β 50 ng/ml has demonstrated effective early and late apoptosis in as short a time period as 6 h. The experimental model used demonstrated 1.5 fold higher production of NO, 1.2 fold increased oxidative stress and lower mitochondrial membrane potential as compared to the positive control used. Hence the above model can be easily used for assessment and screening of drugs that can prevent apoptosis of beta cells and stop progression of type 1 diabetes.

Nitric oxide and nitrous oxide production in anaerobic/anoxic nitrite-denitrifying phosphorus removal process: effect of phosphorus concentration.

Nitric oxide (NO) and nitrous oxide (N2O) production in biological nutrient removal has been studied widely due to the strong negative effects on the environment. Nitrite-denitrifying phosphorus removal (N-DPR), as a significant source of NO and N2O production, has received great attention. However, the mechanism of NO and N2O production at different phosphorus concentrations is not well understood. Therefore, this study was conducted to investigate the effect of phosphorus concentration on pollutant removal, as well as NO and N2O production during the N-DPR process. The results showed that the phosphorus removal efficiency was improved with the increase of phosphorus concentration, which is caused by the enrichment of denitrifying phosphorus accumulating organisms (DPAOs) at high phosphorus concentration. High NO production was observed at phosphorus concentration of 0.5 mg L-1, which is mainly attributed to the slow recovery of reductase activity and low abundance of DPAOs. The maximal N2O accumulation of 31.45 mg L-1 was also achieved at phosphorus concentration of 0.5 mg L-1. The possible reason is that fewer poly-β-hydroxyalkanoates (PHAs) were synthesized by glycogen accumulating organisms (GAOs) at low phosphorus concentration, which could intensify the electron competition among different reductases. In addition, free nitrous acid (FNA) inhibition was another significant reason for high N2O production.

Insights on the Larvicidal Mechanism of Action of Fractions and Compounds from Aerial Parts of Helicteres velutina K. Schum against Aedes aegypti L.

Viral diseases transmitted by the female Aedes aegypti L. are considered a major public health problem. The aerial parts of Helicteres velutina K. Schum (Sterculiaceae) have demonstrated potential insecticidal and larvicidal activity against this vector. The objective of this research was to investigate the mechanisms of action involved in the larvicidal activity of this species. The cytotoxicity activity of H. velutina fractions and compounds of crude ethanolic extract of the aerial parts of this species was assessed by using fluorescence microscopy and propidium iodide staining. In addition, the production of nitric oxide (NO) and hemocyte recruitment were checked after different periods of exposure. The fluorescence microscopy revealed an increasing in larvae cell necrosis for the dichloromethane fraction, 7,4′-di-O-methyl-8-O-sulphate flavone and hexane fraction (15.4, 11.0, and 7.0%, respectively). The tiliroside did not show necrotic cells, which showed the same result as that seen in the negative control. The NO concentration in hemolymph after 24 h exposure was significantly greater for the dichloromethane fraction and the 7,4′-di-O-methyl-8-O-sulphate flavone (123.8 and 56.2 µM, respectively) when compared to the hexane fraction and tiliroside (10.8 and 8.3 µM, respectively). The presence of plasmocytes only in the dichloromethane fraction and 7,4′-di-O-methyl-8-O-sulphate flavone treatments suggest that these would be the hemocytes responsible for the highest NO production, acting as a defense agent. Our results showed that the larvicidal activity developed by H. velutina compounds is related to its hemocyte necrotizing activity and alteration in NO production.

Photobiomodulation (PBM) regulates nitric oxide (NO) production by peripheral blood mononuclear cells (PBMC) isolated from Multiple Sclerosis (MS) patients

Abstract Multiple sclerosis (MS) is an autoimmune disease characterized by myelin destruction, neurodegeneration and mitochondrial dysfunction. High production of nitric oxide (NO) by iNOS contributes to the generation of reactive nitrogen species, leading to increased nitrosative stress and mitochondrial dysfunction linked to MS progression. Current therapies focus on the suppression of the immune system in early stages, but they are not effective in chronic stages where nitrosative stress takes control over pathogenic mechanisms. Thus, new therapies that reduce nitrosative stress and offer neuroprotection at any stage are needed. Photobiomodulation (PBM) has demonstrated efficacy to improve neurodegenerative disorders. Our lab showed that PBM at 670nm reduced clinical severity in the C57BL/6 mouse experimental autoimmune encephalomyelitis model of MS by down-regulation of nitrosative stress, apoptosis, and pro-inflammatory cytokines, and up-regulation of anti-inflammatory cytokines. Likewise, PBM modulated cytokine expression by peripheral blood mononuclear cells (PBMC) and CD4+ T cells isolated from MS patients. Here we show that PBM reduces NO production by PBMC from MS patients. PBMC were isolated by gradient centrifugation, activated with Phytohaemagglutinin (PHA) and received PBM treatment. As surrogate for NO, nitrite production in supernatants was measured by fluorometry. The most effective PBM doses at decreasing nitrite were 830nm (10J/cm2) and pulsed 640nm (3J/cm2). The expression of Interferon-γ correlated with nitrite expression at these same doses. PBM effect was dependent on the disease modifying drug that subjects were taking. The results show the potential of PBM on NO regulation in MS patients.

Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Molecular Pathways Involved in Aerobic Exercise Training Enhance Vascular Relaxation.

The beneficial effects of exercise training on the cardiovascular system are well known. Because our knowledge of exercise-induced vascular function is still limited, we aimed to uncover the molecular mechanisms conditioning the improved vascular relaxation in muscular arteries. Male Wistar-Kyoto rats with the same ability to run on a treadmill after maximal exercise tests were allocated to the following two groups: trained (Tr) (treadmill, 50%-60% of maximal capacity, 5 d·wk) and untrained (UnTr). After 13 wk, the femoral arteries were harvested and used for functional, structural, and molecular analyses. Acetylcholine (ACh)-induced relaxation and nitric oxide (NO) production were enhanced in arteries from Tr rats compared with UnTr rats. Tr arteries exhibited reduced microRNA (miRNA)-124a expression (whose target is caveolin-1), increased the density of caveolae aligned along the sarcolemma and reduced ACh-induced relaxation in the presence of methyl-β-cyclodextrin, which disrupts caveolae. Higher endothelial NO synthase (eNOS) expression with lower miRNA-155 expression and the posttranslational modification of eNOS (phosphorylation of stimulatory Ser1177 and dephosphorylation of inhibitory Thr495) by the PI3-kinase/Akt1/2/3 pathway also contributed to the higher NO production induced by exercise training. Furthermore, increased Cu/Zn- and extracellular-superoxide dismutase expression and enhanced effects of their pharmacological scavenger activity on the ACh-induced response were observed in Tr arteries. The results of the present study provide a molecular basis for exercise-induced NO bioavailability in healthy femoral arteries. Increased caveolae domain and eNOS expression/activity in Tr arteries are associated with downregulation of miRNA-124a and -155, as well as are involved with higher antioxidant defense, subsequently inducing a favorable endothelium-dependent milieu in Tr arteries.

Altered composition of high-lipid diet may generate reactive oxygen species by disturbing the balance of antioxidant and free radicals.

Background In the present era, obesity is increasing rapidly, and high dietary intake of lipid could be a noteworthy risk factor for the occasion of obesity, as well as nonalcoholic fatty liver disease, which is the independent risk factor for type 2 diabetes and cardiovascular disease. For a long time, high-lipid diet (HLD) in "fast food" is turning into part of our everyday life. So, we were interested in fulfilling the paucity of studies by means of preliminary evaluation of these three alternative doses of HLD on a rat model and elucidating the possible mechanism of these effects and divulging the most alarming dose. Methods Thirty-two rats were taken, and of these, 24 were fed with HLD in three distinctive compositions of edible coconut oil and vanaspati ghee in a ratio of 2:3, 3:2 and 1:1 (n = 8), orally through gavage at a dose of 10 mL/kg body weight for a period of 28 days, whereas the other eight were selected to comprise the control group. Results After completion of the experiment, followed by analysis of data it was revealed that hyperlipidemia with increased liver and cardiac marker enzymes, are associated with hepatocellular injury and cardiac damage. The data also supported increased proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). As oxidative stress parameter increased in both liver and heart, there is also an increased in TNF-α due to an increased expression of inducible nitric oxide (NO) synthase, which led to a high production of NO. Moreover, HLD treatment explicitly weakens reasonability of hepatocytes and cardiomyocytes conceivably through G0/G1 or S stage capture or perhaps by means of enlistment of sub-G0/G1 DNA fragmentation and a sign of apoptosis. Conclusions Based on the outcomes, it tends to be inferred that consequences of the present examination uncovered HLD in combination of 2:3 applies most encouraging systemic damage by reactive oxygen species generation and hyperlipidemia and necroapoptosis of the liver and heart. Hence, outcome of this study may help to formulate health care strategy and warns about the food habit in universal population regarding the use of hydrogenated and saturated fats (vanaspati ghee) in diet.

Effect of combined colchicine-corticosteroid treatment on neutrophil/lymphocyte ratio: a predictive marker in Behçet disease activity.

Behçet's disease (BD) is an auto-immune vasculitis, characterized by episodic inflammation of multiple organs. The neutrophil to lymphocyte ratio (NLR) is used as a marker of inflammation in several diseases nowadays. While nitric oxide (NO) seem to be involved in BD pathogenicity. Our study aims to investigate the NLR as an inflammatory marker of BD activity as well as to evaluate the relationship between the NO production and NLR in Algerian BD patients with different clinical manifestations before and under colchicine + corticosteroid treatment. For this purpose, we evaluated the NLR as the ratio of neutrophil count to lymphocyte count in naïve and treated active BD patients with different clinical manifestations and in inactive ones. Furthermore, we assessed NO production by the Griess' method in the same patients. Additionally, we evaluated in vivo interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels using ELISA. Our results indicate that the NLR and nitrite levels were higher in naïve active BD patients. Interestingly, this high ratio and NO production differed according to the clinical manifestations and was associated with an increased risk of mucocutaneous and vascular involvement. Importantly, in treated BD patients NLR was higher in active patients especially in those with mucocutaneous involvement while increased nitrites levels were regardless of the clinical manifestations studied. Both NLR and NO production decreased in these treated active patients. In addition, IL-4 production differed according to the clinical manifestations studied contrary to the IFN-γ production. Collectively our results suggest that the NLR is a potential marker of BD activity in Algerian patients, predicting the disease severity. Moreover, the positive relationship between the NLR and NO production is related to an increased risk of mucocutaneous lesions and vascular involvement. Thus, the application of these two accessible tools could be benefit for the clinical prognosis and treatment of BD.

Phytochemical Screening, Cytotoxicity and Anti-inflammatory Activities of the Leaf Extracts from Lawsonia inermis of Indian Origin to Explore their Potential for Medicinal Uses.

Lawsonia inermis Linn popularly known as Henna, plays an important role in ayurvedic or natural herbal medicines. The presence of phytoconstituents in henna, that may affect the animal or human health adversely, needs to be elucidated for L. inermis Linn species grown in India. The aim of this research was to perform phytochemical screening, and study cytotoxicity and anti-inflammatory activities to understand the potential of leaves of Lawsonia inermis of Indian origin to provide a way forward for therapeutic use in medicine. We assessed the phytochemical profile for the presence of phytoconstituents (alkaloids, carbohydrates, glycosides, steroids, flavonoids, saponins, tannins, proteins/amino acids and gums/mucilage) in various extracts of the plant leaves. The extracts were further purified by column chromatography for the isolation of plant constituents and monitored by TLC, analyzed by Fourier transform infrared FT-IR spectroscopy, H1NMR, and GC-MS analysis. Fractions were assessed for cytotoxicity and anti-inflammatory properties at various concentrations. We assessed the anti-inflammatory activity by nitric oxide production in various leaf extracts determined by Griess assay. All the spectral results suggest that the compounds from the extract contain an aromatic nucleus and OH group along with the methoxy group, allyl as well as vinyl group. Fractions of chloroform/methanolic (7:3) leaf extract of Lawsonia inermis confirmed the presence of the two constituents i.e. fraxetin and 1(3H)-isobenzofuranone. We observed a significant difference in cytotoxicity at higher concentrations in methanol and chloroform: methanol (8:2) leaf extracts (p>0.05), we could not find any significant differences amongst other leaf extracts at different concentrations. Some leaf extracts have potential cytotoxic activity on Vero cells. Reducing the chloroform concentration during extraction decreases the cytotoxic effect on cells. Nitric oxide levels decreased from 1000 μg/ml concentration to lower concentrations with varying degrees. Overall the highest nitric oxide production by CHCl3 (70%)/ MeOH (30%) was observed amongst various fractions at different concentrations. Phytochemical screening and the study of cytotoxicity and anti-inflammatory activities highlight the potential of leaves of the plant to provide a way further for their use in medicine. Fraxetin 1(3H) and isobenzofuranone structures were confirmed in fractions of CHCl3 (70%)/ MeOH (30%) extract as potent constituents. Some leaf extracts have potential cytotoxic activity on Vero cells. Reducing the chloroform concentration during extraction, it decreases the cytotoxic effect on cells. The cytotoxicity studies indicate the presence of cytotoxic compounds in some of these extracts, warranting research for the fabrication of suitable formulations comprising these constituents to reduce dose/toxicity for beneficial effects of the plant components.

Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte.

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host.OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated.METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry.FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines.MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Inorganic nitrite increases the susceptibility of tilapia (Oreochromis niloticus) leucocytes to Streptococcus agalactiae

Deteriorating water quality, especially from high concentrations of nitrite, is currently largely blamed for disease outbreaks in farmed tilapia (Oreochromis niloticus). In this study, the underlying mechanism of nitrite on the susceptibility of tilapia leucocytes to Streptococcus agalactiae (S. agalactiae) was studied. We found that a high dose of heat-killed S. agalactiae decreased tilapia leucocytes cell viability, whereas nitrite decreased the cell viability of leucocytes exposed to a low dose of bacteria. Bacterial challenge increased the production of nitric oxide (NO), whereas nitrite and bacteria coexposure caused higher NO production than nitrite or bacterial exposure alone. Cell viability increased after elimination of NO, and negative correlations existed between cell viability and the NO content, suggesting that nitrite increased the susceptibility of the leucocytes against S. agalactiae was NO-dependent. For a more comprehensive understanding of the mechanism of nitrite affecting disease resistance in tilapia leucocytes, an RNA-Seq-based transcriptome was generated. The results showed that 6173 transcripts were differently expressed, and the differentially expressed transcripts (DETs) of the bacterial group, nitrite group and bacteria-nitrite co-treatment group compared to the control group were selected for GO and KEGG analyses. The DETs in the bacterial group and bacteria-nitrite cotreatment group were highly involved with the membrane component, signal transduction, and immune responses. KEGG analysis showed that the protein processing in the endoplasmic reticulum and the AMPK signaling pathway, which are related to autophagy, were significantly enriched in the cotreatment group but not in bacterial group. In addition, the mRNA expression of ten DETs and several autophagy and apoptosis related genes validated by q-PCR showed the high reliability of the RNA-seq. Taken together, the results of this study suggest that nitrite may increase the susceptibility of tilapia leucocytes to S. agalactiae by generating excess NO to affect the autophagy and apoptosis process.

Chicken lines divergently selected on feather pecking differ in immune characteristics

It is crucial to identify whether relations between immune characteristics and damaging behaviors in production animals exist, as these behaviors reduce animal welfare and productivity. Feather pecking (FP) is a damaging behavior in chickens, which involves hens pecking and pulling at feathers of conspecifics. To further identify relationships between the immune system and FP we characterized high FP (HFP) and low FP (LFP) selection lines with regard to nitric oxide (NO) production by monocytes, specific antibody (SpAb) titers, natural (auto)antibody (N(A)Ab) titers and immune cell subsets. NO production by monocytes was measured as indicator for innate pro-inflammatory immune functioning, SpAb titers were measured as part of the adaptive immune system and N(A)Ab titers were measured as they play an essential role in both innate and adaptive immunity. Immune cell subsets were measured to identify whether differences in immune characteristics were reflected by differences in the relative abundance of immune cell subsets.Divergent selection on FP affected NO production by monocytes, SpAb and N(A)Ab titers, but did not affect immune cell subsets. The HFP line showed higher NO production by monocytes and higher IgG N(A)Ab titers compared to the LFP line. Furthermore the HFP line tended to have lower IgM NAAb titers, but higher IgM and IgG SpAb titers compared to the LFP line. Thus, divergent selection on FP affects the innate and adaptive immune system, where the HFP line seems to have a more responsive immune system compared to the LFP line. Although causation cannot be established in the present study, it is clear that relationships between the immune system and FP exist. Therefore, it is important to take these relationships into account when selecting on behavioral or immunological traits.

Investigation on the role of temperature in macrophage responses

Abstract This study investigated temperature-associated changes in macrophage activation and metabolism across species. Macrophages were isolated from C57BL/6J mice, broiler chicken spleens, and rainbow trout head kidneys, treated with lipopolysaccharide (LPS) and nitric oxide (NO) responses measured. Mouse macrophage NO production was similar at 37°C (normal body temp.) and 35°C. However at 33°C and 28°C responses dropped notably. At 39°C, Mouse bone marrow-derived macrophages (BMDM) and mouse cell line, RAW264.7, showed higher NO production versus 37°C. In chicken splenic macrophages (CSM) LPS-induced NO responses were similar at 41°C (normal for avian) 37°C, and 39°C. A mild fever of 42°C had a large stimulatory effect on NO production compared to 41°C. During infection, trout induce fever by swimming to warmer water. We found NO responses in LPS-treated head kidney macrophages to be higher at 15, 28, and 37°C than at 4°C. Total protein synthesis was evaluated via fluorescence. Without LPS stimulation, peritoneal resident mouse macrophages (PRM) from young mice showed similar protein synthesis at 37°C and 39°C. In both young and aged LPS-stimulated PRM, significantly higher protein synthesis was observed at 39°C compared to 37°C, though the increase was smaller in aged PRM. However, this effect was not uniform across all tissues. LPS stimulated young (BMDM) showed an increased protein synthesis at 37°C but cells from aged did not. A further increase in LPS stimulation was observed at 39°C. Consistent with fever increasing protein synthesis, LPS-stimulated CSM and chicken cell line HTC, showed higher protein synthesis at 42°C compared with 41°C.

The 12-HHT/BLT2/NO Axis Is Associated to the Wound Healing and Skin Condition in Different Glycaemic States.

Type 2 diabetes affects over 340 million people worldwide. This condition can go unnoticed and undiagnosed for years, leading to a late stage where high glycaemia produces complications such as delayed wound healing. Studies have shown that 12-HHT through BLT2, accelerates keratinocyte migration and wound healing. Additionally, evidence has shown the role of nitric oxide as a pro-regenerative mediator, which is decreased in diabetes. Our main goal was to study the association between the 12-HHT/BLT2 axis and the nitric oxide production in wound healing under different glycaemia conditions. For that purpose, we used in vivo and in vitro models. Our results show that the skin from diabetic mice showed reduced BLT2 and iNOS mRNA, TEER, 12-HHT, nitrites, and tight junction levels, accompanied by higher MMP9 mRNA levels. Furthermore, a positive correlation between BLT2 mRNA and nitrites was observed. In vitro, HaCaT-BLT2 cells showed higher nitric oxide and tight junction levels, and reduced MMP9 mRNA levels, compared to mock-keratinocytes under low and high glucose condition. The wound healing capacity was associated with higher nitric oxide production and was affected by the NOS inhibition. We suggest that the BLT2 expression improves the keratinocyte response to hyperglycaemia, associated with the production of nitric oxide.

Valproic acid promotes a decrease in mycobacterial survival by enhancing nitric oxide production in macrophages stimulated with IFN-γ

Tuberculosis is one of the leading causes of mortality worldwide, it is caused by Mycobacterium tuberculosis (Mtb), a bacteria that employs several strategies to evade the host immune response. For instance, Mtb interferes with the overexpression of class II transactivator (CIITA) in macrophages exposed to IFN-γ by inhibiting histone acetylation at its promoter, which can be reverted by the histone deacetylase inhibitor (HDACi) sodium butyrate. In this work, we evaluated whether a different HDACi, valproic acid (VPA), could revert the inhibition of gene expression induced by Mtb. J774 macrophages treated with VPA and IFN-γ unexpectedly induced a higher expression of the inducible nitric oxide synthase and a higher production of nitric oxide when exposed to the 19 kDa lipoprotein of Mtb or the whole bacteria. However, VPA was unable to revert the inhibition of CIITA expression induced by the 19 kDa lipoprotein of Mtb. Finally, macrophages infected with Mtb and treated with VPA and IFN-γ showed a significant reduction in intracellular bacteria. Our findings suggest a new therapeutic potential of VPA for the treatment of tuberculosis.

Solid–state fermentation of germinated black bean (Rhynchosia nulubilis) using Lactobacillus pentosus SC65 and its immunostimulatory effect

Germinated black bean (Rhynchosia nulubilis) (GRN) has several beneficial health effects, including immunostimulation. To enhance the immunostimulatory effect of GRN, it was fermented with lactic acid bacteria (LAB). A total of 57 strains of LAB that showed good growth on GRN were isolated from various fermented foods and vegetables. Among them, Lactobacillus pentosus SC65 was selected as a starter culture for GRN fermentation. Optimum solid–state fermentation conditions of GRN with L. pentosus SC65 (GRN–SC65) were: water content of 40% (v/w), fermentation time of 24 h, fermentation temperature of 30 °C, and inoculation volume of 5% (v/w). RAW264.7 cells treated with hot water extract of GRN–SC65 showed high nitric oxide production and phagocytic activity, indicating high immunostimulatory activity of GRN–SC65.