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Sample records for arginase

  1. Helicobacter pylori arginase mutant colonizes arginase Ⅱ knockout mice

    Institute of Scientific and Technical Information of China (English)

    Songhee H Kim; Melanie L Langford; Jean-Luc Boucher; Traci L Testerman; David J McGee

    2011-01-01

    AIM: To investigate the role of host and bacterial argi-nases in the colonization of mice by Helicobacter pylori (H. Pylori).METHODS: H. Pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. Pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. Pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase Ⅱ, since this enzyme is expressed in the stomach, and H. Pylori has previously been shown to induce the expres-sion of murine gastric arginase Ⅱ. To test this hypoth-esis, wild-type and arginase (rocF) mutant H. Pylori strain SS1 were inoculated into arginase Ⅱ knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase Ⅱ knock-out mice. Moreover, feeding arginase Ⅱ knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase Ⅰactivity in several tissues, did not block the ability of the rocF mutant H. Pylori to colonize. In con-trast, BEC poorly inhibited H. Pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. Pylori urease is neither bacterial arginase nor host arginase Ⅱ; instead, either residual host arginase Ⅰor agmatinase is probably responsible.

  2. Role of arginase in vessel wall remodeling

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    William eDurante

    2013-05-01

    Full Text Available Arginase metabolizes the semi-essential amino acid L-arginine to L-ornithine and urea. There are two distinct isoforms of arginase, arginase I and II, which are encoded by separate genes and display differences in tissue distribution, subcellular localization, and molecular regulation. Blood vessels express both arginase I and II but their distribution appears to be cell-, vessel-, and species-specific. Both isoforms of arginase are induced by numerous pathologic stimuli and contribute to vascular cell dysfunction and vessel wall remodeling in several diseases. Clinical and experimental studies have documented increases in the expression and/or activity of arginase I or II in blood vessels following arterial injury and in pulmonary and arterial hypertension, aging, and atherosclerosis. Significantly, pharmacological inhibition or genetic ablation of arginase in animals ameliorates abnormalities in vascular cells and normalizes blood vessel architecture and function in all of these pathological states. The detrimental effect of arginase in vascular remodeling is attributable to its ability to stimulate vascular smooth muscle cell and endothelial cell proliferation, and collagen deposition by promoting the synthesis of polyamines and L-proline, respectively. In addition, arginase adversely impacts arterial remodeling by directing macrophages towards an inflammatory phenotype. Moreover, the proliferative, fibrotic, and inflammatory actions of arginase in the vasculature are further amplified by its capacity to inhibit nitric oxide synthesis by competing with nitric oxide synthase for substrate, L-arginine. Pharmacologic or molecular approaches targeting specific isoforms of arginase represent a promising strategy in treating obstructive fibroproliferative vascular disease.

  3. Arginase 1 and arginase 2 variations associate with asthma, asthma severity and beta(2) agonist and steroid response

    NARCIS (Netherlands)

    Vonk, Judith M.; Postma, Dirkje S.; Maarsingh, Harm; Bruinenberg, Marcel; Koppelman, Gerard H.; Meurs, Herman

    2010-01-01

    Rationale Arginase probably plays an important role in asthma development, severity and progression. Polymorphisms in arginase 1 and arginase 2 genes have been associated with childhood asthma and FEV1 reversibility to beta(2) agonists. Objectives We investigated the association between arginase 1 a

  4. Contribution of arginase to manganese metabolism of Aspergillus niger.

    Science.gov (United States)

    Keni, Sarita; Punekar, Narayan S

    2016-02-01

    Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (ΔXCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.

  5. Evolutionary roots of arginase expression and regulation

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    Jolanta Maria Dzik

    2014-11-01

    Full Text Available Two main types of macrophage functions are known: classical (M1, producing nitric oxide, NO, and M2, in which arginase activity is primarily expressed. Ornithine, the product of arginase, is a substrate for synthesis of polyamines and collagen, important for growth and ontogeny of animals. M2 macrophages, expressing high level of mitochondrial arginase, have been implicated in promoting cell division and deposition of collagen during ontogeny and wound repair. Arginase expression is the default mode of tissue macrophages, but can also be amplified by signals, such as IL4/13 or TGF-β that accelerates wound healing and tissue repair. In worms, the induction of collagen gene is coupled with induction of immune response genes, both depending on the same TGF-β-like pathway. This suggests that the main function of M2 heal type macrophages is originally connected with the TGF-β superfamily of proteins, which are involved in regulation of tissue and organ differentiation in embryogenesis. Excretory-secretory products of metazoan parasites are able to induce M2 type of macrophage responses promoting wound healing without participation of Th2 cytokines IL4/IL13. The expression of arginase in lower animals can be induced by the presence of parasite antigens and TGF-β signals leading to collagen synthesis. This also means that the main proteins, which, in primitive metazoans, are involved in regulation of tissue and organ differentiation in embryogenesis are produced by innate immunity. The signaling function of NO is known already from the sponge stage of animal evolution. The cytotoxic role of NO molecule appeared later, as documented in immunity of marine mollusks and some insects. This implies that the M2-wound healing promoting function predates the defensive role of NO, a characteristic of M1 macrophages. Understanding when and how the M1 and M2 activities came to be in animals is useful for understanding how macrophage immunity, and immune

  6. Erythrocyte arginase activity as an indicator of lead exposure

    OpenAIRE

    Fukumoto, K.; Karai, I; NISHIKAWA, Y.; Horiguchi, S

    1983-01-01

    ABSTRACT A semi-automated method has been developed for the determination of the arginase activity of erythrocytes using dried blood spots, which are easy to prepare on site in a factory for later laboratory analysis. The mean arginase activity of erythrocytes in 49 men occupationally exposed to lead was 62·9 IU/g·Hb (SD, 14·4 IU/g·Hb); in 45 men not exposed to lead the mean was 44·6 IU/g·Hb (SD, 11·6 IU/g·Hb). A significantly higher mean arginase activity was found in the specimens from lead...

  7. Development of novel arginase inhibitors for therapy of endothelial dysfunction

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    Jochen eSteppan

    2013-09-01

    Full Text Available Endothelial dysfunction and resulting vascular pathology have been identified as an early hallmark of multiple diseases, including diabetes mellitus. One of the major contributors to endothelial dysfunction is a decrease in nitric oxide (NO bioavailability, impaired NO signaling and an increase in the amount of reactive oxygen species (ROS. In the endothelium NO is produced by eNOS (endothelial nitric oxide synthase, for which L-arginine is a substrate. Arginase, an enzyme critical in the urea cycle also metabolizes L-arginine, thereby directly competing with eNOS for their common substrate and constraining its bioavailability for eNOS, thereby compromising NO production. Arginase expression and activity is upregulated in many cardiovascular diseases including ischemia reperfusion injury, hypertension, atherosclerosis, and diabetes mellitus. More importantly, since the 1990s, specific arginase inhibitors such as N-hydroxy-guanidinium or N-hydroxy-nor-L-arginine, and boronic acid derivatives, such as, 2(S-amino-6-boronohexanoic acid, and S-(2-boronoethyl-L-cysteine (BEC, that can bridge the binuclear manganese cluster of arginase have been developed. These highly potent and specific inhibitors can now be used to probe arginase function and thereby modulate the redox milieu of the cell by changing the balance between NO and ROS. Inspired by this success, drug discovery programs have recently led to the identification of α-α-disubstituted amino acid based arginase inhibitors (such as (R-2-amino-6-borono-2-(2-(piperidin-1-ylethylhexanoic acid, that are currently under early investigation as therapeutics. Finally, some investigators concentrate on identification of plant derived compounds with arginase inhibitory capability, such as piceatannol-3'-O-β-D-glucopyranoside (PG. All of these synthesized or naturally derived small molecules may represent novel therapeutics for vascular disease particularly that associated with diabetes.

  8. Inhibition of Human Arginase I by Substrate adn Product Analogues

    Energy Technology Data Exchange (ETDEWEB)

    L Di Costanzo; M Ilies; K Thorn; D Christianson

    2011-12-31

    Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

  9. Probing the Specificity Determinants of Amino Acid Recognition by Arginase

    Energy Technology Data Exchange (ETDEWEB)

    Shishova, E.; Di Costanzo, L; Emig, F; Ash, D; Christianson, D

    2009-01-01

    Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the ?-amino and ?-carboxylate groups) of substrate l-arginine and inhibitors in the active site of arginase I. Specifically, we focus on (1) a water-mediated hydrogen bond between the substrate ?-carboxylate and T135, (2) a direct hydrogen bond between the substrate ?-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate ?-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

  10. Bone marrow cell derived arginase I is the major source of allergen-induced lung arginase but is not required for airway hyperresponsiveness, remodeling and lung inflammatory responses in mice

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    Rothenberg Marc E

    2009-06-01

    Full Text Available Abstract Background Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice. Results In order to test the hypothesis that arginase has a role in allergic airway inflammation we generated arginase I-deficient bone marrow (BM chimeric mice. Following transfer of arginase I-deficient BM into irradiated recipient mice, arginase I expression was not required for hematopoietic reconstitution and baseline immunity. Arginase I deficiency in bone marrow-derived cells decreased allergen-induced lung arginase by 85.8 ± 5.6%. In contrast, arginase II-deficient mice had increased lung arginase activity following allergen challenge to a similar level to wild type mice. BM-derived arginase I was not required for allergen-elicited sensitization, recruitment of inflammatory cells in the lung, and proliferation of cells. Furthermore, allergen-induced airway hyperresponsiveness and collagen deposition were similar in arginase-deficient and wild type mice. Additionally, arginase II-deficient mice respond similarly to their control wild type mice with allergen-induced inflammation, airway hyperresponsiveness, proliferation and collagen deposition. Conclusion Bone marrow cell derived arginase I is the predominant source of allergen-induced lung arginase but is not required for allergen-induced inflammation, airway hyperresponsiveness or collagen deposition.

  11. Purification and Properties of the Constitutive Arginase of Evernia prunastri.

    Science.gov (United States)

    Martín-Falquina, A; Legaz, M E

    1984-12-01

    Constitutive arginase (molecular weight 330,000) 920-fold purified from Evernia prunastri thallus, is activated by putrescine, l-ornithine, and agmatine with K(a) values of 2.7, 1.1, and 5.8 millimolar, respectively. Constitutive arginase is also activated by endogenous l-arginine, reaching its maximum activity at 16 hours of incubation on Tris-HCl (pH 9.15) with a subsequent decrease. Urea behaves as a mixed inhibitor of the enzyme with a K(i) value of 2.6 millimolar. Atranorin and evernic acid behave as in vitro activators of the enzyme; usnic acid does not have any significant effect as activator.

  12. Binding of [alpha, alpha]-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design

    Energy Technology Data Exchange (ETDEWEB)

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W. (MIT); (Episcopal U); (Rutgers); (Drexel); (Penn)

    2011-10-21

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of {alpha},{alpha}-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional {alpha}-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  13. Anesthetic Management of a Pediatric Patient with Arginase Deficiency

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    Abdulkadir Atım

    2011-09-01

    Full Text Available Arginase deficiency is an autosomal recessive disorder of the urea cycle in which a defect in conversion of arginine to urea and ornithine leads to hyperammonemia. Patients with urea cycle disorders may show increased protein catabolism due to inadequate intake of energy, protein and essential amino acids; infections, fever and surgery. A 12-year-old girl with arginase deficiency, ASA II who weighed 40 kg was scheduled for bilateral adductor, quadriceps and gastrocnemius tenotomies. She had mental retardation, spasticity and flexion posture of thelower limbs. Metabolic homeostasis was restored with appropriate diet. Successful anesthetic management allowed the patient to be discharged 48 hours after surgery. Increased levels of arginine and ammonia during or after surgery may lead to serious complications such as hypotension, cerebral edema, convulsions, hypothermia and spasticity. Thus special attention must be given to metabolic homeostasis and nutrition of the patients with arginase deficiency in the perioperative period. Primary goals should be to minimize stress levels by effective anxiolysis, provide an adequate amount of protein-free energy with proper fluid management and to obtain an effective preemptive and postoperative analgesia. In addition to a high level of knowledge, successful anesthesia requires professional communication among nursing staff, dietitians, pediatric metabolism specialist, surgeon and anesthesiologist.

  14. Citrulline Supplementation Improves Organ Perfusion and Arginine Availability under Conditions with Enhanced Arginase Activity

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    Karolina A.P. Wijnands

    2015-06-01

    Full Text Available Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with l-arginine supplementation exhibited less consistent results; however, l-citrulline, the precursor of l-arginine, may be a promising alternative. In this study, we determined the effects of l-citrulline compared to l-arginine supplementation on arginine-nitric oxide (NO metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each injected intraperitoneally with sterile saline or arginase (1000 IE/mouse with or without being separately injected with l-citrulline or l-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF-imaging or in vivo NO-production with electron spin resonance (ESR spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. l-arginine and l-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that l-citrulline, and not l-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

  15. Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates

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    Spadafora Domenico

    2007-04-01

    Full Text Available Abstract Background Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. Results The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. Conclusion These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

  16. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

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    Patterson John R

    2008-09-01

    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  17. Effects of a chronic l-arginine supplementation on the arginase pathway in aged rats.

    Science.gov (United States)

    Moretto, Johnny; Guglielmetti, Anne-Sophie; Tournier-Nappey, Maude; Martin, Hélène; Prigent-Tessier, Anne; Marie, Christine; Demougeot, Céline

    2017-04-01

    While ageing is frequently associated with l-arginine deficiency, clinical and experimental studies provided controversial data on the interest of a chronic l-arginine supplementation with beneficial, no or even deleterious effects. It was hypothesized that these discrepancies might relate to a deviation of l-arginine metabolism towards production of l-ornithine rather than nitric oxide as a result of age-induced increase in arginase activity. This study investigated the effect of ageing on arginase activity/expression in target tissues and determined whether l-arginine supplementation modulated the effect of ageing on arginase activity. Arginase activity and expression were measured in the heart, vessel, brain, lung, kidney and liver in young rats (3-months old) and aged Wistar rats (22-24-months-old) with or without l-arginine supplementation (2.25% in drinking water for 6weeks). Plasma levels of l-arginine and l-ornithine were quantified in order to calculate the plasma l-arginine/l-ornithine ratio, considered as a reflection of arginase activity. Cardiovascular parameters (blood pressure, heart rate) and aortic vascular reactivity were also studied. Ageing dramatically reduced plasma l-arginine and l-arginine/l-ornithine ratio, decreased liver and kidney arginase activities but did not change activities in other tissues. l-Arginine supplementation normalized plasma l-arginine and l-arginine/l-ornithine ratio, improved endothelial function and decreased systolic blood pressure. These effects were associated with decreased arginase activity in aorta along with no change in the other tissues except in the lung in which activity was increased. A strong mismatch was therefore observed between arginase activity and expression in analyzed tissues. The present study reveals that ageing selectively changes arginase activity in clearance tissues, but does not support a role of the arginase pathway in the potential deleterious effect of the l-arginine supplementation in

  18. Some Properties of Purified and Non-purified Rumen Tissue Arginase in Cattle

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    ERİŞİR, Mine; OZAN, Sema Temizer

    1998-01-01

    Some biochemical properties of purified and non-purified rumen tissue arginase were compared. Homogenization, heating, treatment with aceton, precipitation with ammonium sulfate, dialysis, several centrifugations, gel filtration on sephadex G-200 processes were utilized in the purification procedure of the enzyme. It was found that pre-incubation temperature (60 °C) of arginase and Km (4mM) to its substrate, L-arginine, did not change before and after purification. While pre-incubation peri...

  19. HIF2α–arginase axis is essential for the development of pulmonary hypertension

    OpenAIRE

    Cowburn, Andrew S.; Crosby, Alexi; Macias, David; Branco, Cristina; Colaço, Renato D. D. R.; Southwood, Mark; Toshner, Mark; Crotty Alexander, Laura E.; Morrell, Nicholas W.; Chilvers, Edwin R.; Johnson, Randall S.

    2016-01-01

    The expression of hypoxia-inducible factor (HIF)-2α in pulmonary endothelium of mice influences pulmonary vascular resistance and development of hypoxic pulmonary hypertension (PH) via an arginase-1–dependent mechanism. The HIF-2α:arginase-1 axis influences the homeostatic regulation of nitric oxide synthesis in the lung. Impaired generation of this vasoactive agent contributes to the initial development and vascular remodeling process of PH.

  20. Arginase: the emerging therapeutic target for vascular oxidative stress and inflammation

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    Zhihong eYang

    2013-06-01

    Full Text Available Oxidative stress and inflammation in the vascular wall are essential mechanisms of atherosclerosis and vascular dysfunctions associated with risk factors such as metabolic diseases, aging, hypertension, etc. Evidence has been provided that activation of the vascular endothelial cells in the presence of the risk factors promotes oxidative stress and vascular inflammatory responses, leading to acceleration of atherosclerotic vascular disease. Increasing number of studies from recent years demonstrates that uncoupling of endothelial nitric oxide synthase (eNOS, whereby the enzyme eNOS produces detrimental amount of superoxide anion O2.– instead the vasoprotective nitric oxide (NO., plays a critical role in vascular dysfunction under various pathophysiological conditions and in aging. The mechanisms of eNOS-uncoupling seem multiple and complex. Recent research provides emerging evidence supporting an essential role of increased activity of arginases including arginase-I and arginase-II in causing eNOS-uncoupling, which results in vascular oxidative stress and inflammatory responses, and ultimately leading to vascular diseases. This review article will summarize the most recent findings on the functional roles of arginases in vascular diseases and/or dysfunctions and the underlying mechanisms in relation to oxidative stress and inflammations. Moreover, regulatory mechanisms of arginases in the vasculature are reviewed and the future perspectives of targeting arginases as therapeutic options in vascular diseases are discussed.

  1. Neuroblastoma Arginase Activity Creates an Immunosuppressive Microenvironment That Impairs Autologous and Engineered Immunity.

    Science.gov (United States)

    Mussai, Francis; Egan, Sharon; Hunter, Stuart; Webber, Hannah; Fisher, Jonathan; Wheat, Rachel; McConville, Carmel; Sbirkov, Yordan; Wheeler, Kate; Bendle, Gavin; Petrie, Kevin; Anderson, John; Chesler, Louis; De Santo, Carmela

    2015-08-01

    Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.

  2. DMPD: Regulation of nitric oxide synthesis and apoptosis by arginase and argininerecycling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17513437 Regulation of nitric oxide synthesis and apoptosis by arginase and arginin...erecycling. Mori M. J Nutr. 2007 Jun;137(6 Suppl 2):1616S-1620S. (.png) (.svg) (.html) (.csml) Show Regulation of nitric oxide synthe...sis and apoptosis by arginase and argininerecycling. PubmedID 17513437 Title Regulation of nitric oxide synt...hesis and apoptosis by arginase and argininerecycling. A

  3. Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

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    2005-08-01

    Full Text Available Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.

  4. Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis.

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    Talavera, Maria M; Nuthakki, Sushma; Cui, Hongmei; Jin, Yi; Liu, Yusen; Nelin, Leif D

    2017-01-01

    Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using N(ω) hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

  5. Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis

    Science.gov (United States)

    Talavera, Maria M.; Nuthakki, Sushma; Cui, Hongmei; Jin, Yi; Liu, Yusen; Nelin, Leif D.

    2017-01-01

    Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using Nω hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

  6. Local arginase 1 activity is required for cutaneous wound healing.

    Science.gov (United States)

    Campbell, Laura; Saville, Charis R; Murray, Peter J; Cruickshank, Sheena M; Hardman, Matthew J

    2013-10-01

    Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS(+) cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

  7. Effect of arginase inhibition on pulmonary L-arginine metabolism in murine Pseudomonas pneumonia.

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    Anne Mehl

    Full Text Available RATIONALE: Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. METHODS: Mice (C57BL/6, 8-10 weeks old, female underwent direct tracheal instillation of Pseudomonas (PAO-1-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S-amino-6-boronohexanoic acid (ABH or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. RESULTS: NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001, as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001, the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001. Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001 but increase in L-arginine concentration (p<0.001, L-arginine/ADMA ratio (p<0.001, L-arginine availability for NOS (p<0.001, and NO metabolite concentrations (67.3±5.7 µM, p<0.05. Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015. Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1β, IL-6, MIP-2, KC or TNF-α in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased

  8. Chitosan treatment abrogates hypercholesterolemia-induced erythrocyte’s arginase activation

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    Gamaleldin I. Harisa

    2017-01-01

    Full Text Available This study aimed to evaluate the protective effect of chitosan (CS against hypercholesterolemia (HC induced arginase activation and disruption of nitric oxide (NO biosynthesis using erythrocytes as cellular model. Human erythrocytes were isolated and classified into eight groups. Next, cells were treated with l-arginine (l-ARG, Nω-nitro-l-arginine methyl ester (l-NAME, CS or CS + l-ARG in the presence of normal plasma or cholesterol enriches plasma. Then, erythrocytes were incubated at 37 °C for 24 h. The present results revealed that, HC induced significant increase of cholesterol inclusion into erythrocytes membrane compared to control. Moreover, HC caused significant decrease in nitric oxide synthase (NOS activity similar to l-NAME; however, arginase activity and arginase/NOS ratio significantly increased compared to control. On contrast, treatment of HC with, l-arginine, CS or CS plus l-arginine prevents HC induced cholesterol loading into erythrocytes membrane, NOS inhibition and arginase activation. This study suggested that CS could be protective agent against HC induced disruption of erythrocyte’s oxidative status and arginase activation.

  9. Arginase induction and activation during ischemia and reperfusion and functional consequences for the heart

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    Klaus-Dieter eSchlüter

    2015-03-01

    Full Text Available Induction and activation of arginase is among the fastest responses of the heart to ischemic events. Induction of arginase expression and enzyme activation under ischemic conditions shifts arginine consumption from nitric oxide formation (NO to the formation of ornithine and urea. In the heart such a switch in substrate utilisation reduces the impact of the NO/cGMP-pathway on cardiac function that requires intact electromechanical coupling but at the same time it induces ornithine-dependent pathways such as the polyamine metabolism. Both effects significantly reduce the recovery of heart function during reperfusion and thereby limits the success of reperfusion strategies. In this context, changes in arginine consumption trigger cardiac remodelling in an unfavourable way and increases the risk of arrhythmia, specifically in the initial post-ischemic period in which arginase activity is dominating. However, during the entire ischemic period arginase activation might be a meaningful adaptation that is specifically relevant for reperfusion following prolonged ischemic periods. Therefore, a precise understanding about the underlying mechanism that leads to arginase induction as well as of it’s mechanistic impact on post-ischemic hearts is required for optimizing reperfusion strategies. In this review we will summarize our current understanding of these processes and give an outlook about possible treatment options for the future.

  10. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  11. Selective endothelial overexpression of arginase II induces endothelial dysfunction and hypertension and enhances atherosclerosis in mice.

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    Boris L Vaisman

    Full Text Available Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis.Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE -/- mice, hArgII mice had increased aortic atherosclerotic lesions.We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.

  12. An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

    NARCIS (Netherlands)

    Romagnoli, G.; Verhoeven, M.D.; Mans, R.; Fleury Rey, Y.; Bel-Rhlid, R.; Van den Broek, M.; Maleki Seifar, R.; Ten Pierick, A.; Thompson, M.; Müller, V.; Wahl, S.A.; Pronk, J.T.; Daran, J.M.

    2014-01-01

    Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were clon

  13. Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

    Science.gov (United States)

    Pedrosa, M M; Legaz, M E

    1995-04-01

    Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

  14. Inhibitory effect of mycoplasma-released arginase. Activity in mixed-lymphocyte and tumour cell cultures

    DEFF Research Database (Denmark)

    Claesson, M H; Tscherning, T; Nissen, Mogens Holst

    1990-01-01

    Non-fermenting mycoplasma species deplete culture media for arginine through arginase activity linked to their arginine deiminase pathway, resulting in proliferation arrest and cell death in mycoplasma-contaminated cell cultures. The presence of only 2-3 Mycoplasma (M.) arginini-contaminated T...... inhibition can be reversed by addition of excess arginine to the culture medium. Antisera raised against non-fermenting, but not against fermenting, mycoplasma species block the inhibitory effect of MAE. SDS-PAGE separation of MAE disclosed a broad band at 60 kDa which contained arginase activity when...

  15. An Engineered Arginase FC Protein Inhibits Tumor Growth In Vitro and In Vivo

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    Lihua Li

    2013-01-01

    Full Text Available Arginine is a semiessential amino acid required for the growth of melanoma and hepatocellular carcinoma, and the enzymatic removal of arginine by pegylated arginine deiminase (ADI or arginase is being tested clinically. Here, we report a genetically engineered arginase FC fusion protein exhibiting a prolonged half-life and enhanced efficacy. The use of this enzyme to treat different tumor lines both inhibited cell proliferation and impaired cellular migration in vitro and in vivo. Our data reinforce the hypothesis that nutritional depletion is a key strategy for cancer treatment.

  16. TIPE2 negatively regulates inflammation by switching arginine metabolism from nitric oxide synthase to arginase.

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    Yunwei Lou

    Full Text Available TIPE2, the tumor necrosis factor (TNF-alpha-induced protein 8-like 2 (TNFAIP8L2, plays an essential role in maintaining immune homeostasis. It is highly expressed in macrophages and negatively regulates inflammation through inhibiting Toll-like receptor signaling. In this paper, we utilized RAW264.7 cells stably transfected with a TIPE2 expression plasmid, as well as TIPE2-deficient macrophages to study the roles of TIPE2 in LPS-induced nitric oxide (NO and urea production. The results showed that TIPE2-deficiency significantly upregulated the levels of iNOS expression and NO production in LPS-stimulated macrophages, but decreased mRNA levels of arginase I and urea production. However, TIPE2 overexpression in macrophages was capable of downregulating protein levels of LPS-induced iNOS and NO, but generated greater levels of arginase I and urea production. Furthermore, TIPE2-/- mice had higher iNOS protein levels in lung and liver and higher plasma NO concentrations, but lower levels of liver arginase I compared to LPS-treated WT controls. Interestingly, significant increases in IκB degradation and phosphorylation of JNK, p38, and IκB were observed in TIPE2-deficient macrophages following LPS challenge. These results strongly suggest that TIPE2 plays an important role in shifting L-arginase metabolism from production of NO to urea, during host inflammatory response.

  17. Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity

    NARCIS (Netherlands)

    Meurs, Herman; Hamer, M.A M; Pethe, S; Vadon-Le Goff, S; Boucher, J.-L; Zaagsma, Hans

    2000-01-01

    1 Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes f

  18. Recombinant human arginase I immobilized on gold and silver nanoparticles: preparation and properties

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    Natalia Stasyuk

    2011-07-01

    Full Text Available Metal nanoparticles (NPs, such as gold (Au and silver (Ag, are important for chemistry, physics, and biology due to their unique optical, electrical, and photothermal properties. Such NPs are widely used for immobilization of various bioactive substances, including peptides, enzymes, antibodies and DNA. The synthesis of silver and gold nanoparticles was carried out by reduction of silver nitrate by glucose and reduction of tetrachloroauric acid by sodium citrate, respectively. The size and structure of the AgNPs and AuNPs were characterized using TEM, AFM and XRD methods. The average size of the AgNPs and AuNPs was between 8 and 15 nm. Recombinant arginase I was immobilized using the carbodiimidepentafluorophenol method on the surface of NPs functionalized with ω-mercaptohexadecanoic acid. It was shown that recombinant human liver arginase I isolated from the yeast Hansenula polymorpha maintains satisfactory stability after immobilization on both NPs. The immobilized arginase retained 40% of its activity on the surface of AuNPs and 25% on AgNPs compared to the free arginase after storage at +4 ºC during 25 days. The immobilized enzyme can be used for assay of arginine in pharmaceuticals, in food products and in blood.

  19. Augmentation of arginase Ⅱ expression in the human endometrial epithelium in the secretory phase.

    Science.gov (United States)

    Tajima, Makiko; Harada, Tatsuya; Ishikawa, Tomonori; Iwahara, Yuki; Kubota, Toshiro

    2012-12-03

    L-arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to urea and L-ornithine. L-Ornithine is the principal precursor for the production of polyamines and L-proline, which are required for cell proliferation and collagen synthesis. Endothelial NOS is expressed in the human endometrial glandular epithelium, but the expression and physiological roles of arginase in the human endometrium are not clear. The objective of this study was to investigate the expression and distribution patterns of arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) in the human endometrium by using immunohistochemistry, reverse transcription-polymerase chain reaction (RTPCR), and western blotting. A-Ⅰ and A-Ⅱ were detected by immunohistochemistry in human endometrial epithelial cells during the proliferative and secretory phases of the menstrual cycle. RT-PCR showed that A-Ⅰ and A-Ⅱ mRNA were expressed in human endometrial tissue. Western blotting analysis results showed the expression of A-Ⅱ protein. Immunohistochemistry and western blotting results showed that expression levels of A-Ⅱ were significantly higher in the secretory phase than in the proliferative phase. Increased A-Ⅱ levels in the secretory phase may be responsible for endometrial growth by increasing polyamines and proline products.

  20. Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

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    Amatullah Hajera

    2011-02-01

    Full Text Available Abstract Background Arginase overexpression contributes to airways hyperresponsiveness (AHR in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR. Methods To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA and challenged with nebulized PBS (OVA/PBS or OVA (OVA/OVA for three consecutive days (sub-acute model or 12 weeks (chronic model, which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3, or HEPA-filtered air (FA, for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization. Results Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models. Conclusions This study demonstrates that arginase is upregulated following environmental exposures in murine models of

  1. Transcriptional profiling of gastric epithelial cells infected with wild type or arginase-deficient Helicobacter pylori

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    Kim Songhee H

    2012-08-01

    Full Text Available Abstract Background Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators. Results Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type Helicobacter pylori strain 26695, an arginase mutant (rocF- or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction. Conclusions We conclude that H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.

  2. A protective effect of the laminated layer on Echinococcus granulosus survival dependent on upregulation of host arginase.

    Science.gov (United States)

    Amri, Manel; Touil-Boukoffa, Chafia

    2015-09-01

    The role of nitric oxide (NO) in host defense against Echinococcus granulosus larvae was previously reported. However, NO production by NOS2 (inducible NO synthase) is counteracted by the expression of Arginase. In the present study, our aim is to evaluate the involvement of the laminated layer (external layer of parasitic cyst) in Arginase induction and the protoscoleces (living and infective part of the cyst) survival. Our in vitro results indicate that this cystic compound increases the Arginase activity in macrophages. Moreover, C-type lectin receptors (CLRs) with specificity for mannan and the TGF-β are implicated in this effect as shown after adding Mannan and Anti-TGFβ. Interestingly, the laminated layer increases protoscoleces survival in macrophages-parasite co-cultures. Our results indicate that the laminated layer protects E. granulosus against the NOS2 protective response through Arginase pathway, a hallmark of M2 macrophages.

  3. Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

    Science.gov (United States)

    Broman, K; Lauwers, N; Stalon, V; Wiame, J M

    1978-09-01

    Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.

  4. Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase?

    Science.gov (United States)

    Wildy, P; Gell, P G; Rhodes, J; Newton, A

    1982-01-01

    Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus. PMID:6286497

  5. Effect of Two Ginger Varieties on Arginase Activity in Hypercholesterolemic Rats.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Oboh, Ganiyu; Ademiluyi, Adedayo Oluwaseun; Boligon, Aline Augusti; Athayde, Margareth Linde

    2016-04-01

    Recently, ginger has been used in traditional Chinese medicine as an herbal therapy for treating several cardiovascular diseases, however, information on its mechanism of action is limited. The present study assessed the effect of two ginger varieties (Zingiber officinale and Curcuma longa) on the arginase activity, atherogenic index, levels of liver thiobarbituric acid reactive substances (TBARSs), and plasma lipids in rats fed with a high-cholesterol (2%) diet for 14 days. Following the treatment period, it was found that feeding a high-cholesterol diet to rats caused significant (p ginger and turmeric (2% and 4%) caused significant (p ginger and turmeric) inhibited arginase activity and prevented hypercholesterolemia in rats that received a high-cholesterol diet. Therefore, these activities of ginger and turmeric represent possible mechanisms underlying its use in herbal medicine to treat several cardiovascular diseases.

  6. Arginase Flavonoid Anti-Leishmanial in Silico Inhibitors Flagged against Anti-Targets

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    Sanja Glisic

    2016-05-01

    Full Text Available Arginase, a drug target for the treatment of leishmaniasis, is involved in the biosynthesis of polyamines. Flavonoids are interesting natural compounds found in many foods and some of them may inhibit this enzyme. The MetIDB database containing 5667 compounds was screened using an EIIP/AQVN filter and 3D QSAR to find the most promising candidate compounds. In addition, these top hits were screened in silico versus human arginase and an anti-target battery consisting of cytochromes P450 2a6, 2c9, 3a4, sulfotransferase, and the pregnane-X-receptor in order to flag their possible interactions with these proteins involved in the metabolism of substances. The resulting compounds may have promise to be further developed for the treatment of leishmaniasis.

  7. Purification and Properties of the Constitutive Arginase of Evernia prunastri1

    Science.gov (United States)

    Martín-Falquina, Angeles; Legaz, María Estrella

    1984-01-01

    Constitutive arginase (molecular weight 330,000) 920-fold purified from Evernia prunastri thallus, is activated by putrescine, l-ornithine, and agmatine with Ka values of 2.7, 1.1, and 5.8 millimolar, respectively. Constitutive arginase is also activated by endogenous l-arginine, reaching its maximum activity at 16 hours of incubation on Tris-HCl (pH 9.15) with a subsequent decrease. Urea behaves as a mixed inhibitor of the enzyme with a Ki value of 2.6 millimolar. Atranorin and evernic acid behave as in vitro activators of the enzyme; usnic acid does not have any significant effect as activator. PMID:16663950

  8. A natural compound macelignan protects midbrain dopaminergic neurons from inflammatory degeneration via microglial arginase-1 expression.

    Science.gov (United States)

    Kiyofuji, Kana; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Mishima, Satoshi; Katsuki, Hiroshi

    2015-08-01

    Inflammatory events involving activated microglia have been recognized to play an important role in pathogenesis of various neurodegenerative disorders including Parkinson disease. Compounds regulating activation profiles of microglia may provide therapeutic benefits for Parkinson disease characterized by degeneration of midbrain dopaminergic neurons. Here we examined the effect of macelignan, a compound derived from nutmeg, on inflammatory degeneration of midbrain dopaminergic neurons. Treatment of midbrain slice cultures with interferon (IFN)-γ and lipopolysaccharide (LPS) caused a substantial decrease in viable dopaminergic neurons and an increase in nitric oxide (NO) production indicated by extracellular nitrite accumulation. Application of macelignan (10 μM) concomitantly with LPS prevented the loss of dopaminergic neurons. Besides nitrite accumulation, up-regulation of inducible NO synthase protein expression in response to IFN-γ/LPS was confirmed by Western blotting, and immunohistochemical examination revealed expression of inducible NO synthase in a subpopulation of Iba-1-poitive microglia. However, macelignan did not affect any of these NO-related parameters. On the other hand, macelignan promoted expression of arginase-1 in midbrain slice cultures irrespective of the presence or the absence of IFN-γ/LPS treatment. Arginase-1 expression was mainly localized in a subpopulation of Iba-1-positive cells. Importantly, the neuroprotective effect of macelignan was antagonized by N(ω)-hydroxy-nor-L-arginine, a specific arginase inhibitor. The neuroprotective effect of macelignan was also prevented by GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Overall, these results indicate that macelignan, a compound with PPARγ agonist activity, can provide neuroprotective effect on dopaminergic neurons in an arginase-dependent but NO-independent manner.

  9. Candida albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions

    Science.gov (United States)

    MacCallum, Donna M.; Brown, Gordon D.

    2017-01-01

    ABSTRACT   The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival. PMID:28119468

  10. Influence of βS-Globin Haplotypes and Hydroxyurea on Arginase I Levels in Sickle Cell Disease.

    Science.gov (United States)

    Moreira, J A; Machado, R P G; Laurentino, M R; Lemes, Romelia Pinheiro Gonçalves; Barbosa, M C; Santos, T E; Bandeira, I C J; Martins, A M C

    2016-01-01

    Introduction. Sickle cell disease (SCD) is characterized by hemoglobin S homozygosity, leading to hemolysis and vasoocclusion. The hemolysis releases arginase I, an enzyme that decreases the bioavailability of nitric oxide, worsening the symptoms. The different SCD haplotypes are related to clinical symptoms and varied hemoglobin F (HbF) concentration. The aim of this study was to evaluate the impact of the βS gene haplotypes and HbF concentration on arginase I levels in SCD patients. Methods. Fifty SCD adult patients were enrolled in the study and 20 blood donors composed the control group. Arginase I was measured by ELISA. The βS haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Statistical analyses were performed with GraphPad Prism program and the significance level was p haplotypes groups showed a difference between the Bantu/Bantu × Bantu/Benin groups; Bantu/Bantu × Benin/Benin, independent of HU dosage. An inverse correlation with the arginase I levels and HbF concentration was observed. Conclusion. The results support the hypothesis that arginase I is associated with HbF concentration, also measured indirectly by the association with haplotypes.

  11. Modulation of the arginase pathway in the context of microbial pathogenesis: a metabolic enzyme moonlighting as an immune modulator.

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    Priyanka Das

    Full Text Available Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology.

  12. Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

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    Manuel Modolell

    Full Text Available The balance between T helper (Th 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

  13. Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline

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    Ruffieux Jean

    2009-03-01

    Full Text Available Abstract Background Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells. Methods Human endothelial cells were isolated from umbilical veins and stimulated with TNFα (10 ng/ml for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1, intercellular adhesion molecule-1 (ICAM-1, and E-selectin were assessed by immunoblotting. Results The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFα was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl-L-cysteine (BEC had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFα-induced activation of NF-κB, JNK, p38mapk, while it inhibited p70s6k (S6K1 activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFα. Conclusion The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

  14. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus 1

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    Legaz, María Estrella; Vicente, Carlos

    1983-01-01

    Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID:16662821

  15. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus.

    Science.gov (United States)

    Legaz, M E; Vicente, C

    1983-02-01

    Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation.

  16. Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during Giardia lamblia infection.

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    Maloney, Jenny; Keselman, Aleksander; Li, Erqiu; Singer, Steven M

    2015-06-01

    Nitric oxide (NO) has been shown to inhibit Giardia lamblia in vitro and in vivo. This study sought to determine if Giardia infection induces arginase 1 (ARG1) expression in host macrophages to reduce NO production. Stimulations of RAW 264.7 macrophage-like cells with Giardia extract induced arginase activity. Real-time PCR and immunohistochemistry showed increased ARG1 and nitric oxide synthase 2 (NOS2) expression in mouse intestine following infection. Flow cytometry demonstrated increased numbers of macrophages positive for both ARG1 and NOS2 in lamina propria following infection, but there was no evidence of increased expression of ARG1 in these cells.

  17. Role of arginase 1 from myeloid cells in th2-dominated lung inflammation.

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    Luke Barron

    Full Text Available Th2-driven lung inflammation increases Arginase 1 (Arg1 expression in alternatively-activated macrophages (AAMs. AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.

  18. Cationic amino acid transporter-2 regulates immunity by modulating arginase activity.

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    Robert W Thompson

    2008-03-01

    Full Text Available Cationic amino acid transporters (CAT are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/- mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/- mice developed stronger IFN-gamma responses, nitric oxide (NO production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/- mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/- mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.

  19. HIF2α–arginase axis is essential for the development of pulmonary hypertension

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    Cowburn, Andrew S.; Crosby, Alexi; Macias, David; Branco, Cristina; Colaço, Renato D. D. R.; Southwood, Mark; Toshner, Mark; Crotty Alexander, Laura E.; Morrell, Nicholas W.; Chilvers, Edwin R.; Johnson, Randall S.

    2016-01-01

    Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia. PMID:27432976

  20. Secreted arginases from phylogenetically farrelated lichen species act as cross-recognition factors for two different algal cells.

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    Legaz, María-Estrella; Fontaniella, Blanca; Millanes, Ana-María; Carlos, Vicente

    2004-08-01

    Purified arginases secreted from Evernia prunastri and Xanthoria parietina thalli hydrolyze arginine in a Mn2+ -dependent reaction. Ca2+ cannot replace Mn2+, but its addition to reaction mixtures in the presence of Mn2+ significantly inhibited arginase activity. Arginases from both lichen species also show lectin function, binding to the cell wall of both homologous and heterologous algae. Such binding is enhanced by both Ca2+ and Mn2+ and results in cytoagglutination, which is counteracted by alpha-D-galactose. A putative ligand for these lectins consists of a glycosylated urease, the polysaccharide moiety of which is uniquely composed of alpha-D-galactose. Binding of lectins inhibits its enzymatic activity, which is recovered after desorption of the lectin with alpha-D-galactose. Urease is also eluted from arginase-agarose columns by using alpha-D-galactose as eluent. Data demonstrate ligand-dependent retention of the fungal lectin on the algal cell surface and this is consistent with a model of recognition of compatible algae, through which algal cells would form a lichen with a lectin-secreting fungus only when these cells contain the specific ligand for the lectin in their cell walls. This is, lectin binding is used as a mechanism for ensuring specificity in the association.

  1. Association of ADAM33 gene polymorphism and arginase activity with susceptibility to ventilatory impairment in wood dust-exposed workers.

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    Saad-Hussein, A; Thabet, E H; Taha, M M; Shahy, E M; Mahdy-Abdallah, H

    2016-09-01

    ADAM33 represents an important gene of susceptibility for lung function impairment. This work aimed to evaluate the association between genetic polymorphism of ADAM33 at four single nucleotide polymorphisms (T1, T2, S1, and Q1) and arginase activity with respiratory functions impairment in wood workers. The study was done to compare ventilatory functions and arginase activity of 82 wood workers and 81 controls. Genotyping was determined by using the polymerase chain restriction fragment length polymorphism method. Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and peak expiratory flow rate (PEF) of the workers were significantly reduced compared with the controls. T1 single nucleotide polymorphism (SNP) was associated with obvious decline in the FEV1, FVC, and PEF in wood workers, while T2 SNP was associated with decline in FEV1 and PEF. A significant increase in arginase activity was found in T2 and S1 SNPs of the exposed workers. Increase in duration of exposure was correlated with the decline in ventilatory functions. This inverse correlation was significant for pulmonary function indices in AA and GG genotypes of T1 and T2, respectively. Moreover, significance was detected for FVC and FEV1 in AA and GA genotypes of S1 and Q1. A positive correlation between arginase activity and duration of exposure was found to be significant in GG genotype of S1 SNP. An association between ADAM33 gene polymorphism and impaired lung functions was detected in wood dust-exposed workers. Arginase activity may play an associated important role in increasing this impairment in wood workers.

  2. The Increase in Mannose Receptor Recycling Favors Arginase Induction and Trypanosoma Cruzi Survival in Macrophages

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    Vanina V. Garrido, Laura R. Dulgerian, Cinthia C. Stempin, Fabio M. Cerbán

    2011-01-01

    Full Text Available The macrophage mannose receptor (MR is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand biased nitric oxide (NO/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and

  3. Arginase 1 is an innate lymphoid cell-intrinsic metabolic checkpoint controlling type 2 inflammation

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    Monticelli, Laurel A; Buck, Michael D; Flamar, Anne-Laure; Saenz, Steven A; Wojno, Elia D Tait; Yudanin, Naomi A; Osborne, Lisa C; Hepworth, Matthew R; Tran, Sara V; Rodewald, Hans-Reimer; Shah, Hardik; Cross, Justin R; Diamond, Joshua M; Cantu, Edward; Christie, Jason D; Pearce, Erika L; Artis, David

    2016-01-01

    Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair following activation by cell-extrinsic factors including host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme Arginase 1 (Arg1) is a conserved trait of murine and human ILC2s during acute or chronic lung inflammation. Deletion of murine ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics, controlling proliferative capacity and pro-inflammatory functions that promote type 2 inflammation. PMID:27043409

  4. Carbon nanotube-based sensing devices for human Arginase-1 detection

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    S. Baldo

    2016-03-01

    Full Text Available A new carbon nanotube-based device for detection of Arginase 1 (ARG-1 was produced. Multi-walled carbon nanotubes (MWCNTs were deposited between electrodes by dielectrophoresis (DEP in an accurate and reproducible way. This deposition method has the advantages of low cost and room temperature conditions and therefore, can be used on different kinds of substrates (silicon, glass, plastics allowing for large scale production of chemical or biological sensors. Scanning electrical microscope (SEM and electrical characterization have been performed on the biosensors before and after protein exposure. The devices were tested in the present work for the detection of ARG-1. They show high sensitivity and reproducibility, and can be easily and suitably modified to detect other proteins.

  5. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages

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    Meera eRath

    2014-10-01

    Full Text Available Macrophages play a major role in the immune system, both as antimicrobial effector cells and as immunoregulatory cells, which induce, suppress or modulate adaptive immune responses. These key aspects of macrophage biology are fundamentally driven by the phenotype of macrophage arginine metabolism that is prevalent in an evolving or ongoing immune response. M1 macrophages express the enzyme nitric oxide synthase (NOS, which metabolizes arginine to nitric oxide (NO and citrulline. NO can be metabolized to further downstream reactive nitrogen species, while citrulline might be reused for efficient NO synthesis via the citrulline-NO cycle. M2 macrophages are characterized by expression of the enzyme arginase, which hydrolyzes arginine to ornithine and urea. The arginase pathway limits arginine availability for NO synthesis and ornithine itself can further feed into the important downstream pathways of polyamine and proline syntheses, which are important for cellular proliferation and tissue repair. M1 versus M2 polarization leads to opposing outcomes of inflammatory reactions, but depending on the context, M1 and M2 macrophages can be both pro- and antiinflammatory. Notably, M1/M2 macrophage polarization can be driven by microbial infection or innate danger signals without any influence of adaptive immune cells, secondarily driving the T helper (Th1/Th2 polarization of the evolving adaptive immune response. Since both arginine metabolic pathways cross-inhibit each other on the level of the respective arginine break-down products and Th1 and Th2 lymphocytes can drive or amplify macrophage M1/M2 dichotomy via cytokine activation, this forms the basis of a self-sustaining M1/M2 polarization of the whole immune response. Understanding the arginine metabolism of M1/M2 macrophage phenotypes is therefore central to find new possibilities to manipulate immune responses in infection, autoimmune diseases, chronic inflammatory conditions and cancer.

  6. Alternative activation of macrophages and induction of arginase are not components of pathogenesis mediated by Francisella species.

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    Amanda J Griffin

    Full Text Available Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.

  7. Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes

    Science.gov (United States)

    Boitz, Jan M.; Gilroy, Caslin A.; Olenyik, Tamara D.; Paradis, Dustin; Perdeh, Jasmine; Dearman, Kristie; Davis, Madison J.; Yates, Phillip A.; Li, Yuexin; Riscoe, Michael K.; Ullman, Buddy

    2016-01-01

    ABSTRACT Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite. PMID:27795357

  8. Periodontal therapy reduces arginase activity in saliva of patients with chronic periodontitis.

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    Gheren, L W; Cortelli, J R; Rodrigues, E; Holzhausen, M; Saad, W A

    2008-03-01

    This present study evaluated the salivary arginase activity (SAA) in patients with chronic periodontitis and the effect of periodontal therapy on the activity of such enzyme. Thirty-six patients (mean age, 45.97 +/- 14.52), 18 chronic periodontitis subjects (test group), and 18 periodontally healthy individuals (control group) participated in the study. Clinical periodontal examinations included measurements of probing pocket depth (PD), clinical attachment level (CAL), plaque (PI), and gingival (GI) indexes. The test group received periodontal therapy according to individual needs. The saliva sample was collected from all study population at baseline (both groups) and 30 days after periodontal therapy (test group). SAA was determined by measuring the L: -ornithine formation from L-arginine and was expressed as mU/ml. The results showed that the mean values of SAA were statistically different between control and test groups. SAA was about 2.5 times higher in test than control groups. Thirty days after periodontal therapy, enzyme levels were 1.56 times lower than before periodontal therapy. We concluded that SAA is increased in chronic periodontitis subjects when compared to periodontally healthy individuals and that periodontal therapy significantly reduced SAA levels. It was suggested that in the near future, SAA may be used as a salivary marker of periodontal status.

  9. Thermokinetic Study on the Reversible Competitive Inhibition of Bovine Liver Arginase

    Institute of Scientific and Technical Information of China (English)

    XIE, Xiu-Yin(谢修银); WANG, Cun-Xin(汪存信); WANG, Zhi-Yong(王志勇)

    2004-01-01

    A new thermokinetic reduced extent method for studying of the reversible competitive inhibition of single substrate enzyme-catalyzed reactions was proposed in this paper. The reaction that arginase-catalyzed hydrolysis of L-arginine to L-ornithine and urea and the inhibition of this reaction by the product, L-ornithine, and exogenous L-lysine were studied at 37℃ in 40 mmol·L-1 sodium barbitrurate-HCI buffer solution (pH=9.4).Michealis constant Km for arginine and maximum velocity Vm of the reaction were determined to be 5.14 mmol·L-1 and 1.13×10-2 mmol·L-1·s-1,respectively. The product inhibition were constant Kp and inhibitory constant K1 of L-lysine were determined to be 1.18 and 5.6 mmol·L-1 respectively. All the results have hetter repeatability and self-consistency and are in agreement with literature values. This new method using more direct thermal information from the process would give more reliable kinetic information than the traditional initial rate method.

  10. Arginase-1-expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis.

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    John T Pesce

    2009-04-01

    Full Text Available Macrophage-specific expression of Arginase-1 is commonly believed to promote inflammation, fibrosis, and wound healing by enhancing L-proline, polyamine, and Th2 cytokine production. Here, however, we show that macrophage-specific Arg1 functions as an inhibitor of inflammation and fibrosis following infection with the Th2-inducing pathogen Schistosoma mansoni. Although susceptibility to infection was not affected by the conditional deletion of Arg1 in macrophages, Arg1(-/flox;LysMcre mice died at an accelerated rate. The mortality was not due to acute Th1/NOS2-mediated hepatotoxicity or endotoxemia. Instead, granulomatous inflammation, liver fibrosis, and portal hypertension increased in infected Arg1(-/flox;LysMcre mice. Similar findings were obtained with Arg1(flox/flox;Tie2cre mice, which delete Arg1 in all macrophage populations. Production of Th2 cytokines increased in the infected Arg1(-/flox;LysMcre mice, and unlike alternatively activated wild-type macrophages, Arg1(-/flox;LysMcre macrophages failed to inhibit T cell proliferation in vitro, providing an underlying mechanism for the exacerbated Th2 pathology. The suppressive activity of Arg1-expressing macrophages was independent of IL-10 and TGF-beta1. However, when exogenous L-arginine was provided, T cell proliferation was restored, suggesting that Arg1-expressing macrophages deplete arginine, which is required to sustain CD4(+ T cell responses. These data identify Arg1 as the essential suppressive mediator of alternatively activated macrophages (AAM and demonstrate that Arg1-expressing macrophages function as suppressors rather than inducers of Th2-dependent inflammation and fibrosis.

  11. Deprivation of arginine by recombinant human arginase in prostate cancer cells

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    Hsueh Eddy C

    2012-04-01

    Full Text Available Abstract Background Recombinant human arginase (rhArg has been developed for arginine deprivation therapy in cancer, and is currently under clinical investigation. During pre-clinical evaluation, rhArg has exhibited significant anti-proliferative activity in cancer cells deficient in the expression of ornithine carbamoyl transferase (OCT. Interestingly, a variety of cancer cells such as melanoma and prostate cancer deficient in argininosuccinate synthetase (ASS are sensitive to arginine deprivation by arginine deiminase. In this study, we investigated levels of gene expression of OCT and ASS, and the effects of rhArg in human prostate cancer cells: LNCaP (androgen-dependent, PC-3 and DU-145 (both androgen-independent. Results Quantitative real-time PCR showed minimal to absent gene expression of OCT, but ample expression of ASS expression in all 3 cell lines. Cell viability assay after 72-h exposure of rhArg showed all 3 lines had half maximal inhibitory concentration less than or equal to 0.02 U/ml. Addition of ornithine to cell culture media failed to rescue these cells from rhArg-mediated cytotoxicity. Decreased phosphorylation of 4E-BP1, a downstream effector of mammalian target of rapamycin (mTOR, was noted in DU-145 and PC-3 after exposure to rhArg. Moreover, there was no significant apoptosis induction after arginine deprivation by rhArg in all 3 prostate cancer cell lines. Conclusion rhArg causes significant cytotoxicity in LNCaP, DU-145 and PC-3 prostate cancer cells which all demonstrate decreased OCT expression. Inhibition of mTOR manifested by hypophosphorylation of 4E-BP1 suggests autophagy is involved as alternative cell death mechanism. rhArg demonstrates a promising novel agent for prostate cancer treatment.

  12. Arginase 1 mediates increased blood pressure and contributes to vascular endothelial dysfunction in deoxycorticosterone acetate (DOCA-salt hypertension

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    Haroldo A. Toque

    2013-07-01

    Full Text Available Enhanced arginase (ARG activity has been identified as a factor that reduces nitric oxide (NO production and impairs endothelial function in vascular pathologies. Using a gene deletion model, we investigated involvement of arginase isoforms (1 and 2 in hypertension and endothelial dysfunction in a mineralocorticoid-salt mouse model. Hypertension was induced in wild type (WT, partial ARG1+/- knockout (KO and complete ARG2-/- KO mice by uninephrectomy and DOCA-salt treatment for 6-weeks. (Control uninephrectomized mice drank tap water. After 2 wks of DOCA-salt treatment, systolic blood pressure (SBP was increased by ~15 mmHg in all mouse genotypes. SBP continued to rise in DOCA-salt WT and ARG2-/- mice to ~130 mmHg at 5-6 wks, whereas in ARG1+/- mice SBP waned toward control levels by 6 wks (109±4 vs 101±3 mmHg, respectively. DOCA-salt treatment in WT mice increased vascular ARG activity (aorta by 1.5-fold; mesenteric artery (MA by 2.6-fold and protein levels of ARG1 (aorta: 1.49-fold and MA: 1.73-fold versus WT Sham tissues. ARG2 protein increased in WT DOCA MA (by 2.15-fold but not in aorta compared to those of WT Sham tissues. Maximum endothelium-dependent vasorelaxation to acetylcholine was significantly reduced in DOCA-salt WT mice and largely or partially maintained in DOCA ARG1+/- and ARG2-/- mice vs their Sham controls. DOCA-salt augmented contractile responses to phenylephrine in aorta of all mouse genotypes. Additionally, treatment of aorta or MA from WT DOCA mice with arginase inhibitor ABH (100 µM improved endothelium-mediated vasorelaxation. DOCA-salt induced coronary perivascular fibrosis (increased by 2.1-fold in WT was prevented in ARG1+/- and reduced in ARG2-/- mice. In summary, arginase is involved in murine DOCA-salt induced impairment of vascular function and hypertension and may represent a novel target for antihypertensive therapy.

  13. Arginase 2 deletion leads to enhanced M1 macrophage activation and upregulated polyamine metabolism in response to Helicobacter pylori infection.

    Science.gov (United States)

    Hardbower, Dana M; Asim, Mohammad; Murray-Stewart, Tracy; Casero, Robert A; Verriere, Thomas; Lewis, Nuruddeen D; Chaturvedi, Rupesh; Piazuelo, M Blanca; Wilson, Keith T

    2016-10-01

    We reported that arginase 2 (ARG2) deletion results in increased gastritis and decreased bacterial burden during Helicobacter pylori infection in mice. Our studies implicated a potential role for inducible nitric oxide (NO) synthase (NOS2), as Arg2 (-/-) mice exhibited increased NOS2 levels in gastric macrophages, and NO can kill H. pylori. We now bred Arg2 (-/-) to Nos2 (-/-) mice, and infected them with H. pylori. Compared to wild-type mice, both Arg2 (-/-) and Arg2 (-/-) ;Nos2 (-/-) mice exhibited increased gastritis and decreased colonization, the latter indicating that the effect of ARG2 deletion on bacterial burden was not mediated by NO. While Arg2 (-/-) mice demonstrated enhanced M1 macrophage activation, Nos2 (-/-) and Arg2 (-/-) ;Nos2 (-/-) mice did not demonstrate these changes, but exhibited increased CXCL1 and CXCL2 responses. There was an increased expression of the Th1/Th17 cytokines, interferon gamma and interleukin 17, in gastric tissues and splenic T-cells from Arg2 (-/-), but not Nos2 (-/-) or Arg2 (-/-) ;Nos2 (-/-) mice. Gastric tissues from infected Arg2 (-/-) mice demonstrated increased expression of arginase 1, ornithine decarboxylase, adenosylmethionine decarboxylase 1, spermidine/spermine N (1)-acetyltransferase 1, and spermine oxidase, along with increased spermine levels. These data indicate that ARG2 deletion results in compensatory upregulation of gastric polyamine synthesis and catabolism during H. pylori infection, which may contribute to increased gastric inflammation and associated decreased bacterial load. Overall, the finding of this study is that ARG2 contributes to the immune evasion of H. pylori by restricting M1 macrophage activation and polyamine metabolism.

  14. Galectin-1 Reduces Neuroinflammation via Modulation of Nitric Oxide-Arginase Signaling in HIV-1 Transfected Microglia: a Gold Nanoparticle-Galectin-1 "Nanoplex" a Possible Neurotherapeutic?

    Science.gov (United States)

    Aalinkeel, Ravikumar; Mangum, Courtney S; Abou-Jaoude, Eliane; Reynolds, Jessica L; Liu, Maixian; Sundquist, Karin; Parikh, Neil U; Chaves, Lee D; Mammen, Manoj J; Schwartz, Stanley A; Mahajan, Supriya D

    2017-03-01

    Galectins are a family of β-galactoside-binding lectins that are important modulators of homeostasis in the central nervous system (CNS). Galectin-1 is a pivotal regulator of microglia activation that alters the immune balance from neurodegeneration to neuroprotection and could have therapeutic relevance in HIV associated neurocognitive disorders (HAND). We have previously shown that galectin-1 treatment decreased oxidative stress in microglia and hypothesize that the mechanism underlying this phenomenon is the cross regulatory interactions between Nitric oxide (NO) and Arginase I activity in microglia. We induced microglial activation and examined the effect of galectin-1 on the expression of various M1/M2 microglial phenotypic markers. Since, TNF-α is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases, we treated HIV transfected human microglial cell cultures (CHME-5/HIV) with TNF-α followed by treatment with galectin-1, to examine the galectin-1 mediated neuro-modulatory response. Our results show that treatment of CHME-5/HIV microglia with galectin-1 reduced TNF-α induced oxidative stress by ~40%, and also significantly reduced iNOS gene expression and NO production while correspondingly increasing arginase-1, cationic amino acid transporter (CAT-1) gene expression and arginase activity. Galectin-1 treatment results in shifting microglia polarization from M1 toward the beneficial M2 phenotype which may prevent neurodegeneration and promote neuroprotection. Thus, our data suggests that galectin-1 treatment reduces neuroinflammation in the CNS microenvironment via the modulation of the NO-arginase network in microglia and thus could play a neuroprotective role in HAND. Further, the therapeutic potential of galectin-1 could be enhanced by conjugation of galectin-1 onto gold nanoparticles (Au-NP), resulting in a nanogold-galectin-1 (Au-Gal-1) multivalent complex that will have more clinical translational

  15. Lower expression of inducible nitric oxide synthase and higher expression of arginase in rat alveolar macrophages are linked to their susceptibility to Toxoplasma gondii infection.

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    Zhi-Jun Zhao

    Full Text Available Rats are naturally resistant to Toxoplasma gondii infection, particularly the RH strain, while mice are not. Previous studies have demonstrated that inducible nitric oxide synthase (iNOS and arginase-1 of rodent peritoneal macrophages are linked to the mechanism of resistance. As an increasing number of studies on human and animal infections are showing that pulmonary toxoplasmosis is one of the most severe clinical signs from T. gondii infection, we are interested to know whether T. gondii infection in alveolar macrophages of rats is also linked to the levels of iNOS and arginase-1 activity. Our results demonstrate that T. gondii could grow and proliferate in rat alveolar macrophages, both in vitro and in vivo, at levels higher than resistant rat peritoneal macrophages and at comparable levels to sensitive mouse peritoneal macrophages. Lower activity and expression levels of iNOS and higher activity and expression levels of arginase-1 in rat alveolar macrophages were found to be linked to the susceptibility of T. gondii infection in these cells. These novel findings could aid a better understanding of the pathogenesis of clinical pulmonary toxoplasmosis in humans and domestic animals.

  16. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

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    Celia Maria Vieira Vendrame

    2014-01-01

    Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

  17. Inhibition of Leishmania (Leishmania amazonensis and rat arginases by green tea EGCG, (+-catechin and (--epicatechin: a comparative structural analysis of enzyme-inhibitor interactions.

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    Matheus Balduíno Goncalves dos Reis

    Full Text Available Epigallocatechin-3-gallate (EGCG, a dietary polyphenol (flavanol from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+-catechin and (--epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L and rat liver arginase (ARG-1. The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+-catechin and (--epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+-catechin (IC50 = 0.8 µM followed by (--epicatechin (IC50 = 1.8 µM, gallic acid (IC50 = 2.2 µM and EGCG (IC50 = 3.8 µM. Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC50 values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.

  18. Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

    Science.gov (United States)

    Astigiano, Simonetta; Morini, Monica; Damonte, Patrizia; Fraternali Orcioni, Giulio; Cassanello, Michela; Puglisi, Andrea; Noonan, Douglas M; Bronte, Vincenzo; Barbieri, Ottavia

    2015-11-01

    Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.

  19. In vitro studies to assess the antioxidative, radical scavenging and arginase inhibitory potentials of extracts from Artocarpus altilis, Ficus exasperate and Kigelia africana

    Institute of Scientific and Technical Information of China (English)

    Olubukola Oyebimpe Akanni; Solomon Eduviere Owumi; Oluwatosin Adekunle Adaramoye

    2014-01-01

    Objective: To justify the use of Artocarpus altilis (A. altilis), Ficus exasperata (F. exasperata) and Kigelia africana (K. africana) in ethnomedicine for the treatment of several ailments and to evaluate the in vitro antioxidant, radical scavenging and arginase inhibitory potentials of these herbs and compared with catechin (Standard). Methods:The A. altilis, F. exasperata and K. africana showed dose-dependent and significant Antioxidant activities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, hydrogen peroxide (H2O2) and hydroxyl (OH) radicals scavenging methods. The flavonoids and phenolics content, inhibition of arginase activity, Fe2+/ascorbate-induced lipid peroxidation (LPO) and reducing power were also determined.Results:scavenging of DPPH, H2O2 and OH radicals in vitro relative to catechin. The A. altilis and F.exasperata effectively scavenged DPPH radical with IC50 of 593 and 635 µg/mL and, OH with IC50 of 487 and 514 µg/mL, respectively. The DPPH and OH radicals scavenging activities followed the order A. altilis>F. exasperata>K. africana. In addition, A. altilis and F. exasperata significantly (PCatechin>F. exasperata>K. africana at 1000 µg/mL. The A. altilis at 500 and 750 µg/mL significantly (P<0.05) inhibited arginase activity by 63% and 67%, respectively. The flavonoids contents were found to be highest in A. altilis.Conclusions:Extracts of A. altilis and F. exasperata are potent antioxidative agents with strong radical radical scavenging activity and inhibition of lipid peroxidation.

  20. Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control.

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    Barbara A Butcher

    2011-09-01

    Full Text Available The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16 parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18. In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

  1. Deletion of Genes Encoding Arginase Improves Use of "Heavy" Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast.

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    Weronika E Borek

    Full Text Available The use of "heavy" isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of "heavy"-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This "arginine conversion problem" significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when (13C6-arginine (Arg-6 is used for labeling, it is less successful when (13C6(15N4-arginine (Arg-10, a theoretically preferable label, is used. In particular, we find that with this method, "heavy"-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of (13C5(15N2-arginine (Arg-7 in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.

  2. Purification and immobilization of L-arginase from thermotolerant Penicillium chrysogenum KJ185377.1; with unique kinetic properties as thermostable anticancer enzyme.

    Science.gov (United States)

    El-Sayed, Ashraf S; Shindia, Ahmed A; Diab, Ayman A; Rady, Amgad M

    2014-10-18

    L-Arginase, hydrolyzing L-arginine to L-ornithine and urea, is a powerful anticancer, L-arginine-depleting agent, against argininosuccinate synthase expressing tumors. Otherwise, the higher antigenicity and lower thermal stability of this enzyme was the main biochemical hurdles. Since, the intrinsic thermal stability of enzymes follow the physiological temperature of their producer, thus, characterization of L-arginase from thermotolerant Penicillium chrysogenum was the objective of this study. L-Arginase (Arg) was purified to its homogeneity from P. chrysogenum by 10.1-fold, with 37.0 kDa under denaturing PAGE, optimum reaction at 50 °C, pH stability (6.8-7.9), with highest molar ratio of constitutional arginine, glutamic acid, lysine and aspartic acid. The purified enzyme was PEGylated and immobilized on chitosan, with 41.9 and 22.1 % yield of immobilization. At 40 °C, the T1/2 value of free-Arg, PEG-Arg and Chit-Arg was 10.4, 15.6, 20.5 h, respectively. The free-Arg and Chit-Arg have a higher affinity to L-arginine (K m 4.8 mM), while, PEG-Arg affinity was decreased by about 3 fold (K m 15.2 mM). The inhibitory constants to the free and PEG-Arg were relatively similar towards HA and PPG. The IC50 for the free enzyme against HEPG-2 and A549 tumor cells was 0.136 and 0.165 U/ml, comparing to 0.232 and 0.496 U/ml for PEG-Arg, respectively. The in vivo T1/2 to the free Arg and PEG-Arg was 16.4 and 20.4 h, respectively as holo-enzyme. The residual L-arginine level upon using free Arg was 156.9 and 144.5 µM, after 6 and 8 h, respectively, regarding to initials at 253.6 µM, while for Peg-Arg the level of L-arginine was nil till 7 h of initial dosing. The titer of IgG was induced by 10-15 % in response to free-Arg after 28 days comparing to IgG titer for PEG-Arg.

  3. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

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    Preeti Shahi

    2016-01-01

    Full Text Available Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

  4. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

    Science.gov (United States)

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Alterzon-Baumel, Sharon; Hertz, Rivka; Methling, Karen; Lalk, Michael; Ankri, Serge

    2016-01-01

    Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

  5. 金属离子Zn2+对精氨酸酶抑制作用的研究%Investigation on the inhibition against arginase catalyzed reaction by Zn2+

    Institute of Scientific and Technical Information of China (English)

    史竞艳; 王志勇; 刘欲文; 汪存信

    2012-01-01

    The influence of Zn2+ on arginase catalyzed reaction was studied by microcalorimetry at 37°C ,pH = 9. 4,40 mmol · L-1 sodium barbiturate-hydrochloric acid buffer solution. The result indicated that Zn2+ has remarkable inhibition effect on the catalytic activity of arginase and the inhibitory type belongs to the reversible competitive inhibition with the inhibition constants of 6. 85xlCT7 mol · L-1. According to the inhibition type and ion radius,we suggest that the reason for inhibition is the competition of Zn 2+on the active site of arginase and thus inhibits the reaction activity of arginase.%在37℃,pH=9.4,40 mmol·L-1的巴比妥钠-盐酸缓冲体系中,利用微量热法研究了金属离子Zn2+对牛肝精氨酸酶催化L-精氨酸水解反应的影响.实验表明,Zn2+对精氨酸酶催化反应存在着明显抑制作用,其抑制类型为竞争性可逆抑制,并求得抑制常数K1为6.85×10-7 mol·L-1.根据其抑制类型和离子半径,推测Zn2+对精氨酸酶的抑制主要是夺取Mn2+的位置,从而使酶失活.

  6. DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

    Science.gov (United States)

    Slocum, R D; Bitonti, A J; McCann, P P; Feirer, R P

    1988-10-01

    DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [( 3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

  7. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.

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    Schleicher, Ulrike; Paduch, Katrin; Debus, Andrea; Obermeyer, Stephanie; König, Till; Kling, Jessica C; Ribechini, Eliana; Dudziak, Diana; Mougiakakos, Dimitrios; Murray, Peter J; Ostuni, Renato; Körner, Heinrich; Bogdan, Christian

    2016-05-01

    Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  8. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection

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    Ulrike Schleicher

    2016-05-01

    Full Text Available Neutralization or deletion of tumor necrosis factor (TNF causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1 expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO synthase (NOS2 was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  9. Pegylated derivatives of recombinant human arginase (rhArg1 for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC

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    Wheatley Denys N

    2009-04-01

    Full Text Available Abstract Background Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. Methods The preparation of novel polyethylene glycol (PEG derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1. rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. Results Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000 coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. Conclusion Valuable data on the optimization of the pegylation procedure and

  10. 精氨酸酶在大鼠实验性牙周炎牙周组织中的表达变化%Expression of arginase in periodontal tissues of experimental periodontitis in rats

    Institute of Scientific and Technical Information of China (English)

    孙继军; 王爱芹; 高艳

    2012-01-01

    AIM; To investigate the expression of arginase (Arg) in periodontal tissues at different stages of disease progression in a rat experimental periodontitis model. METHODS: Foiirty male Wistar rats were fed with high sucrose diet. Silk was ligated around the cervix of right first maxillary molars to induce periodontitis ( experimental group). The left first maxillary molars remained untreated to serve as the control. Ten rats were sacrificed at the end of week 1,2,4 and 6. Expression of Arg in periodontal tissues at different stages was detected with immunohistochemistry staining and image quantitative analysis. RESULTS; Arg was scarcely expressed in normal periodontal tissues and was mainly expressed in macrophages and endothelial cells in the experimental groups. The Arg expression increased with the ligation time, reached peak at week 4, and decreased afterwards. CONCLUSION; Arg expression is positively re-lated with the degree of periodontal inflammation.%目的:观察精氨酸酶(arginase,Arg)在不同时期、不同严重程度大鼠实验性牙周炎牙周组织中表达的变化,探讨Arg与牙周炎的相关性.方法:选用40只Wistar雄性大鼠,每只大鼠均用丝线结扎右下颌第一磨牙(实验组),左下颌第一磨牙不做处理(对照组),喂以高糖饮食.分别于结扎后1、2、4、6周,经临床和组织学(HE染色)观察确认大鼠牙龈炎、不同程度牙周炎模型建立成功后,取大鼠下颌骨连同受试牙和牙周组织,制备切片,免疫组化染色法检测正常对照组、牙龈炎组、不同程度牙周炎组大鼠牙周组织中Arg的表达,并进行图像定量分析.结果:在正常牙周组织中,偶有表达;而各实验组牙周组织中Arg主要在巨噬细胞和血管内皮细胞中表达,其表达阳性细胞数随炎症程度加重而增加,结扎4周(重度牙周炎)时达到高峰,到慢性牙周炎时期(结扎6周),表达较急性期有所降低,各组间比较,差异均有统计学意义(P<0

  11. 精氨酸酶Ⅰ基因型与慢性牙周炎、冠心病的相关性研究%Relationship of arginase Ⅰprotein genetic polymorphisms with chronic periodontitis and coronary heart disease

    Institute of Scientific and Technical Information of China (English)

    孙继军; 王晶; 张燕; 王青山; 王爱芹

    2016-01-01

    Objective:To investigate the association of arginase Ⅰ(ArgⅠ)rs2781 666 G/T genotype with the susceptibility of mod-erate and severe chronic periodontitis(MSP)and coronary heart disease(CHD).Methods:DNA was obtained from buccal swabs of 50 patients with MSP,46 with CHD,42 with MSP and CHD,and 50 matched healthy controls.Genotypes of rs2781 666 G/T was analyzed by PCR-PFLP method.The genotype distribution in the 4 groups was compared.Results:Higher representation of ArgⅠrs2781 666 al-lele T was found in CHD group than that in the healthy control individuals(P <0.01 ),and there was no statistical differences among the other 3 groups.Conclusion:Allele T of ArgⅠrs2781 666 may be associated with CHD susceptibility.%目的:探讨精氨酸酶Ⅰ(ArgⅠ)rs2781666 G/T 基因型与中重度慢性牙周炎(MSP)、冠心病(CHD)易感性的关系。方法:收集 MSP 患者50例(MSP 组)、CHD 患者46例(CHD 组)、MSP 伴 CHD 患者42例(MSP +CHD 组)与50例同族健康对照组(HC)的颊黏膜拭子,提取 DNA,采用聚合酶反应-限制性片段长度多态性法(PCR-PFLP)对 ArgⅠrs2781666 G/T 位点的基因型进行检测,比较其在4组间检出率的差别。结果:ArgⅠ rs2781666等位基因 T 在 CHD 组中的检出率显著高于健康组,在其余各组间无统计学意义。结论:ArgⅠrs2781666等位基因 T 可能是 CHD 的一个易感基因。

  12. Expression of arginase Ⅰ in peripheral superficial lymph node of HIV-infected individuals%精氨酸酶Ⅰ在HIV感染者外周浅表淋巴结中表达的研究

    Institute of Scientific and Technical Information of China (English)

    张乃春; 黄磊; 王松山; 邓建宁; 卢祥婵; 吴峰耀; 赵敏

    2015-01-01

    目的 检测精氨酸酶Ⅰ (Arg 1)在HIV感染者外周浅表淋巴结中的表达并探究其与HIV/AIDS进展的关系.方法 根据HIV感染者外周血中CD4+T淋巴细胞计数将患者分组,应用免疫组化方法检测HIV感染者与非HIV感染者外周浅表淋巴结中精氨酸酶Ⅰ的表达,用SPSS17.0软件进行统计分析.结果 HIV感染者外周浅表淋巴结中精氨酸酶Ⅰ的表达明显高于非HIV感染者淋巴结(P<0.01).不同外周血CD4+T淋巴细胞计数的HIV感染者淋巴结中精氨酸酶Ⅰ的表达水平不同.CD4+T淋巴细胞计数≥350/ μl与200/μl≤CD4+T淋巴细胞计数<350/μl的HIV感染者淋巴结中精氨酸酶Ⅰ表达水平差异无统计学意义(P>0.05),而外周血CD4+T淋巴细胞计数≥350/μl,200/ μl≥CD4 +T淋巴细胞计数<350/ μl的HIV感染者外周浅表淋巴结中精氨酸酶Ⅰ较CD4+T淋巴细胞计数< 200/μl的AIDS患者高,差异有统计学意义(P<0.05).且外周血CD4+T淋巴细胞与精氨酸酶Ⅰ的表达水平呈负相关性.结论 以上结果提示Arg Ⅰ在外周浅表淋巴结中的高表达,并与AIDS疾病的进展具有相关性.%Objective To investigate the expression of arginase Ⅰ (.Arg Ⅰ) in peripheral superficial lymph nodes of HIV-infected individuals and to explore their correlation with HIV/AIDS disease progression.Methods All the patients were divided to two groups according to the CD4 + T cell counts in peripheral blood,immunohistochemistry was used to detect expression of Arg Ⅰ in peripheral superficial lymph node of non HIV-infected and HIV-infected individuals.The data were statistically analyzed with SPSS17.0.Results Levels of Arg Ⅰ expression in peripheral superficial lymph node of HIV-infected individuals were higher than those of non HIV-infected lymph nodes (P < 0.01).The expressions of Arg Ⅰ in patients with AIDS in different CD4 + T lymphocyte counts were distinct.Levels of Arg Ⅰ expression in peripheral superficial lymph

  13. Arginase: A Novel Proliferative Determinant in Prostate Cancer

    Science.gov (United States)

    2007-08-01

    TABLE 1. Comparison of Polyamine Levels in Human Prostate Cancer Cell Lines* Cell Line Putrescine Spermidine Spermine...as the aminopropyl donor to synthesize spermidine and spermine from putrescine (Hayashi et al 1997, Kramer et al 1988). Real-time RT-PCR analysis on... putrescine and spermidine levels in these cell lines (Fig. 4). 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Antizyme AdoMetDC R el at iv e

  14. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E

    2006-01-01

    regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here...... a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure...

  15. The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies.

    NARCIS (Netherlands)

    Wagemaker, M.J.M.; Welboren, W.; Drift, C. van der; Jetten, M.S.M.; Griensven, L.J.L.D. van; Camp, H.J.M. op den

    2005-01-01

    An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the clonin

  16. Arginase-1 is a more sensitive marker than HepPar-1 and AFP in differential diagnosis of hepatocellular carcinoma from nonhepatocellular carcinoma.

    Science.gov (United States)

    Sang, Wei; Zhang, Wei; Cui, Wenli; Li, Xinxia; Abulajiang, Gulinar; Li, Qiaoxin

    2015-05-01

    Distinguishing hepatocellular carcinoma (HCC) from metastatic tumors is a challenging issue, especially in differential diagnosis between poorly differentiated HCC and metastasis tumors. Expression of Arg-1, HepPar-1, and α-fetoprotein (AFP) in 78 cases of HCC, 34 cases of metastatic tumors, and 228 cases of nonhepatocellular tumors of surgical specimens is measured by immunohistochemistry. Arg-1 immunoreactivity was detected in 75 of 78 (96.1 %) cases of HCC, whereas HepPar-1 and AFP immunoreactivity was detected in 63 of 78 (80.7 %) and 40 of 78 (51.3 %) cases of HCC, respectively. HepPar-1 and AFP expression was observed in three of 34 (8.8 %) cases and one of 34 (2.9 %) cases of metastatic tumors, respectively. In contrast, Arg-1 expression was absent in all 34 (0 %) cases of metastatic tumors. The sensitivity, specificity, positive predictive value, and negative predictive value of Arg-1 in distinguishing HCC from metastatic tumors and nonhepatocellular tumors are 96.1, 99.6, 98.7, and 98.8 % compared with 80.7, 92.0, 75.0, and 94.1 % for HepPar-1 and 51.3, 97.7, 87.0, and 87.1 % for AFP, respectively. Arg-1 is a more sensitive and better specific marker for HCC compared with HepPar-1 and AFP, indicating that Arg-1 can be easily applied in distinguishing HCC from metastatic tumors.

  17. Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients

    OpenAIRE

    2014-01-01

    Background Low concentrations of nitric oxide (NO) are necessary for the biology and physiology of spermatozoa, but high levels of NO are toxic and have negative effects on sperm functions. Although several studies have considered the relationship between infertility and semen NO concentrations, no study on the effects of asthenospermia treatments such as oral zinc supplementation on concentrations of NO, which are important in fertility, has been reported. Studies have shown that oral zinc s...

  18. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice.

    Directory of Open Access Journals (Sweden)

    Jian Liu

    Full Text Available Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE and Bruch's membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b(+ cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF (+ Arg-1(+ myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF (+ Arg-1(+ phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain.

  19. Evolutionary conservation of alternative activation of macrophages: structural and functional characterization of arginase 1 and 2 in carp (Cyprinus carpio L.)

    NARCIS (Netherlands)

    Joerink, M.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2006-01-01

    Classically activated macrophages (caMF) play an important role in type-I immune responses and alternatively activated macrophages (aaMF) function in type-II immune responses. While the classical activation of fish macrophages has been well described, the existence of aaMF has not yet been described

  20. 日本血吸虫精氨酸酶基因启动子序列的扩增及序列分析%Amplification and Sequence Analysis of Schistosoma japonicum Arginase Novel Gene's Promoter Sequence

    Institute of Scientific and Technical Information of China (English)

    李孜; 余新炳; 吴忠道

    2005-01-01

    目的获取日本血吸虫(Sj)精氨酸酶(ARG)新基因的DNA编码序列及其启动子序列,实验验证ARG基因编码序列的完整性.方法 以日本血吸虫成虫 DNA做模板,PCR扩增 ARG基因编码区的DNA序列并测序;根据Sj ARG基因已扩增的 DNA序列设计2条巢式引物,用TaKaRa LA Taq PCR Cloning in vitro Kit,扩增 Sj ARG基因的启动子序列;对已扩增得到的序列进行 TATA盒的寻找以分析启动子序列的位置,实验验证我们曾获取的 Sj ARG新基因编码序列的完整性. 结果 扩增得到长约 1 000bp ARG基因 DNA序列 , Sj ARG基因 DNA序列内没有内含子,与 cDNA序列完全一致.对启动子序列扩增后,得到一略大于 250bp的序列,测序后分析发现其中有 36bp与 ARG基因 DNA序列的 5′端重叠.因此 ,将精氨酸酶基因的 DNA序列又向前延伸了 221bp,与前面得到的序列拼接后得到一条 1 486bp的总序列,将该序列在 NCBI上进行 ORF的寻找发现其最长的 ORF达 1 164bp,起始密码子在第 156位,终止密码子在第 1 319位 ,该起始密码子前 32位为 TATA盒的位置.因此,确定该 Sj ARG基因全长 cDNA编码序列长应为 1 164bp.结论 成功扩增获得了日本血吸虫精氨酸酶基因编码区的 DNA序列,其不含有内含子;并扩增得到了该基因的启动子序列,从而获得了 Sj ARG基因真正的全长 cDNA序列,为进一步的功能鉴定奠定了基础.

  1. Arginine metabolism in wounds

    Energy Technology Data Exchange (ETDEWEB)

    Albina, J.E.; Mills, C.D.; Barbul, A.; Thirkill, C.E.; Henry, W.L. Jr.; Mastrofrancesco, B.; Caldwell, M.D.

    1988-04-01

    Arginine metabolism in wounds was investigated in the rat in 1) lambda-carrageenan-wounded skeletal muscle, 2) Schilling chambers, and 3) subcutaneous polyvinyl alcohol sponges. All showed decreased arginine and elevated ornithine contents and high arginase activity. Arginase could be brought to the wound by macrophages, which were found to contain arginase activity. However, arginase was expressed by macrophages only after cell lysis and no arginase was released by viable macrophages in vitro. Thus the extracellular arginase of wounds may derive from dead macrophages within the injured tissue. Wound and peritoneal macrophages exhibited arginase deiminase activity as demonstrated by the conversion of (guanido-/sup 14/C)arginine to radiolabeled citrulline during culture, the inhibition of this reaction by formamidinium acetate, and the lack of prokaryotic contamination of the cultures. These findings and the known metabolic fates of the products of arginase and arginine deiminase in the cellular populations of the wound suggest the possibility of cooperativity among cells for the production of substrates for collagen synthesis.

  2. The cell recognition model in chlorolichens involving a fungal lectin binding to an algal ligand can be extended to cyanolichens.

    Science.gov (United States)

    Vivas, M; Sacristán, M; Legaz, M E; Vicente, C

    2010-07-01

    Leptogium corniculatum, a cyanolichen containing Nostoc as photobiont, produces and secretes arginase to culture medium containing arginine. This secreted arginase was pre-purified by affinity chromatography on beads of activated agarose to which a polygalactosylated urease, purified from Evernia prunastri, was attached. Arginase was eluted from the beads with 50 mm alpha-d-galactose. The eluted arginase binds preferentially to the cell surface of Nostoc isolated from this lichen thallus, although it is also able to bind, to some extent, to the cell surface of the chlorobiont isolated from E. prunastri. Previous studies in chlorolichens have shown that a fungal lectin that develops subsidiary arginase activity can be a factor in recognition of compatible algal cells through binding to a polygalactosylated urease, which acts as a lectin ligand in the algal cell wall. Our experiments demonstrate that this model can now be extended to cyanolichens.

  3. Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina.

    Science.gov (United States)

    Díaz, Eva-María; Sacristán, Mara; Legaz, María-Estrella; Vicente, Carlos

    2009-07-01

    Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM alpha-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.

  4. Glycoproteins from sugarcane plants regulate cell polarity of Ustilago scitaminea teliospores.

    Science.gov (United States)

    Millanes, Ana-María; Fontaniella, Blanca; Legaz, María-Estrella; Vicente, Carlos

    2005-03-01

    Saccharum officinarum, cv. Mayarí, is a variety of sugarcane resistant to smut disease caused by Ustilago scitaminea. Sugarcane naturally produces glycoproteins that accumulate in the parenchymatous cells of stalks. These glycoproteins contain a heterofructan as polysaccharide moiety. The concentration of these glycoproteins clearly increases after inoculation of sugarcane plants with smut teliospores, although major symptoms of disease are not observed. These glycoproteins induce homotypic adhesion and inhibit teliospore germination. When glycoproteins from healthy, non-inoculated plants are fractionated, they inhibit actin capping, which occurs before teliospore germination. However, inoculation of smut teliospores induce glycoprotein fractions that promote teliospore polarity and are different from those obtained from healthy plants. These fractions exhibit arginase activity, which is strongly enhanced in inoculated plants. Arginase from healthy plants binds to cell wall teliospores and it is completely desorpted by sucrose, but only 50% of arginase activity from inoculated plants is desorpted by the disaccharide. The data presented herein are consistent with a model of excess arginase entry into teliospores. Arginase synthesized by sugarcane plants as a response to the experimental infection would increase the synthesis of putrescine, which impedes polarization at concentration values higher than 0.05 mM. However, smut teliospores seem to be able to change the pattern of glycoprotein production by sugarcane, thereby promoting the synthesis of different glycoproteins that activate polarization after binding to their cell wall ligand.

  5. L-arginine metabolism in mitochondria isolated from the liver of Antarctic fish Notothenia rossii and Notothenia neglecta

    Directory of Open Access Journals (Sweden)

    Edson Rodrigues

    2006-09-01

    Full Text Available The arginase tissue distribution, the biochemical properties of the argininolytic system and the subcellular localization of the enzymes carbamoylphosphate synthetase, ornithinecarbamoyl transferase, glutamine synthetase and arginase in Antarctic fish, N. neglecta and N. rossii were the main aims of the present work. The tissue with highest argininolytic activity was the kidney distal portion amounting as much as four times the specific activity of the hepatic tissue. Arginase and ornithine carbamoyltransferase were found as mitochondrial enzymes, while glutamine synthetase and carbamoylphosphate synthetase were found as cytosolic enzymes. Argininolytic assays with isolated mitochondria gave values of Kmapp for the hydrolysis of arginine 2 to 3.5 times higher than the values found for the Km with mitochondrial extracts. The effect of Mn2+ on the argininolytic activity displayed by isolated mitochondria and mitochondrial extracts, in reaction conditions near the physiological ones showed that membranes were fundamentally involved in the control of L-arginine metabolism.

  6. Effect of arsenite on urea production by long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    Sierra-Santoyo, A; Hernández, A; López, M L; Mendoza-Figueroa, T

    1996-01-01

    Urea cycle is a hepatic metabolic pathway involving five enzymes and several intermediary metabolites and can be altered by different chemicals. To investigate the effect of arsenic, an ubiquitous hepatotoxic agent, on urea production we exposed long-term cultures of adult rat hepatocytes, which produce urea, to 1.33 and 6.67 microM arsenite for 2 weeks. In cultures exposed to 6.67 microM, urea production decreased 60-70% and cellular arginase activity decreased 30, 70 and 85% after 4, 7 and 14 days of exposure, respectively. The arginase activity released to the medium increased significantly after 4, 7 and 14 days, with a maximum value after 7 days of exposure that was 27-fold higher than that of the untreated controls. The total arginase activity also decreased 35, 52 and 82% after 4, 7 and 14 days of exposure and protein content decreased 57 and 65% after 7 and 14 days of exposure, respectively. Exposure to 6.67 microM arsenite also produced accumulation of intracytoplasmic lipid droplets, vacuolizations and enlargement of the intercellular spaces. On the other hand, exposure of hepatocytes to 1.33 microM arsenite caused an initial decrease of 20% in urea production, did not change cellular, released and total arginase activity and cellular protein content and produced accumulation of intracytoplasmic lipid droplets. These results show that long-term exposure of cultured rat hepatocytes to 6.67 microM arsenite decreases urea production, cellular and total arginase activity and protein content and increases the release of arginase into the culture medium. These alterations could be useful markers of hepatotoxicity in in vitro assays.

  7. Domain Modeling: NP_000036.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available rdination interactions in the binuclear manganese cluster. Resolution 1.60 A. p3f80...NP_000036.2 chr6 (S)-2-amino-6-nitrohexanoic acid binds to human arginase I through multiple nitro-metal coo

  8. Domain Modeling: NP_001163.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available h multiple nitro-metal coordination interactions in the binuclear manganese cluster. Resolution 1.60 A. p3f8...NP_001163.1 chr14 (S)-2-amino-6-nitrohexanoic acid binds to human arginase I throug

  9. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats’ Penile Tissues

    Directory of Open Access Journals (Sweden)

    Ganiyu Oboh

    2015-01-01

    Full Text Available This study was designed to determine the antioxidant properties and inhibitory effects of extract from Moringa oleifera leaves on angiotensin-I-converting enzyme (ACE and arginase activities in vitro. The extract was prepared and phenolic (total phenols and flavonoid contents, radical (nitric oxide (NO, hydroxyl (OH scavenging abilities, and Fe2+-chelating ability were assessed. Characterization of the phenolic constituents was done via high performance liquid chromatography-diode array detection (HPLC-DAD analysis. Furthermore, the effects of the extract on Fe2+-induced MDA production in rats’ penile tissue homogenate as well as its action on ACE and arginase activities were also determined. The extract scavenged NO∗, OH∗, chelated Fe2+, and inhibited MDA production in a dose-dependent pattern with IC50 values of 1.36, 0.52, and 0.38 mg/mL and 194.23 µg/mL, respectively. Gallic acid, chlorogenic acid, quercetin, and kaempferol were the most abundant phenolic compounds identified in the leaf extract. The extract also inhibited ACE and arginase activities in a dose-dependent pattern and their IC50 values were 303.03 and 159.59 µg/mL, respectively. The phenolic contents, inhibition of ACE, arginase, and Fe2+-induced MDA production, and radical (OH∗, NO∗ scavenging and Fe2+-chelating abilities could be some of the possible mechanisms by which M. oleifera leaves could be used in the treatment and/or management of erectile dysfunction.

  10. Fluorometric enzymatic assay of L-arginine

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  11. Protection against herbivores

    Energy Technology Data Exchange (ETDEWEB)

    Howe, Gregg A; Chen, Hui

    2014-10-28

    The present invention relates to genes, proteins and methods comprising molecules that alter amino acid levels. In one embodiment, the present invention relates to altering guanidino substrate hydrolysis activities in plants, arthropods and microorganisms using molecules within the arginase family and other molecules that alter an amino acid levels. In ones embodiment, the present invention relates to altering threonine substrate deamination and dehydration activities in plants, arthropods and microorganisms using molecules within the threonine deaminase family and other molecules that alter amino acid levels. In one embodiment, the present invention relates to using genes, proteins and methods comprising arginase or threonine deaminase for altering the pathophysiology of plants, arthropods and microorganisms. In a preferred embodiment, the present invention relates to altering guanidino substrate hydrolysis activity in plants, arthropods, and microorganisms using arginase. In another preferred embodiment, the invention relates to altering threonine substrated deamination and dehydration activity in plants, arthropods, and microorganisms using threonine deaminase. In some embodiments, the invention related to overexpression and increased activity of arginase, threonine deaminase and a proteinase inhibitor.

  12. Arginine Deficiency Causes Runting in the Suckling Period by Selectively Activating the Stress Kinase GCN2

    NARCIS (Netherlands)

    V. van Marion; S. Sankaranarayanan; C. de Theije; P. van Dijk; P. Lindsey; M.C. Lamers; H.P. Harding; D. Ron; W.H. Lamers; S.E. Koehler

    2011-01-01

    Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-ki

  13. A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells

    NARCIS (Netherlands)

    G. Mondanelli (Giada); R. Bianchi (Roberta); M.T. Pallotta (Maria Teresa); C. Orabona (Ciriana); E. Albini (Elisa); A. Iacono (Alberta); M.L. Belladonna (Maria Laura); C. Vacca (Carmine); F. Fallarino (Francesca); A. Macchiarulo (Antonio); S. Ugel (Stefano); V. Bronte (Vincenzo); F. Gevi (Federica); L. Zolla (Lello); A.P. Verhaar (Auke); M.P. Peppelenbosch (Maikel); E.M.C. Mazza (Emilia Maria Cristina); S. Bicciato (Silvio); Y. Laouar (Yasmina); L. Santambrogio (Laura); P. Puccetti (Paolo); C. Volpi (Claudia); U. Grohmann (Ursula)

    2016-01-01

    textabstractArginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity

  14. Disease: H00186 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00186 Hyperargininemia Hyperargininemia is an autosomal recessive disorder caused ...207800 PMID:16602094 Scaglia F, Lee B Clinical, biochemical, and molecular spectrum of hyperargininemia due ...to arginase I deficiency. Am J Med Genet C Semin Med Genet 142C:113-20 (2006) PMID:15694174 Crombez EA, Cederbaum SD Hype

  15. Th1/Th2 Cytokines: An Easy Model to Study Gene Expression in Immune Cells

    Science.gov (United States)

    Moran, Jose M.; Gonzalez-Polo, Rosa A.; Soler, German; Fuentes, Jose M.

    2006-01-01

    This report describes a laboratory exercise that was incorporated into a Cell Biology and Molecular Biology advanced course. The exercise was made for a class size with eight students and was designed to reinforce the understanding of basic molecular biology techniques. Students used the techniques of reverse transcription and arginase activity…

  16. Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality

    Directory of Open Access Journals (Sweden)

    S. Eskiocak

    2006-05-01

    Full Text Available It has been reported that mental stress causes abnormality of spermiogram parameters. We investigated the effect of psychological stress on the L-arginine-nitric oxide (NO pathway. Semen samples were collected from 29 healthy fourth semester medical students just before (stress and 3 months after (non-stress the final examinations. Psychological stress was measured by the State Anxiety Inventory questionnaire. After standard semen analysis, arginase activity and NO concentration were measured spectrophotometrically in the seminal plasma. Measurements were made in duplicate. During the stress period, sperm concentration (41.28 ± 3.70 vs 77.62 ± 7.13 x 10(6/mL, rapid progressive motility of spermatozoa (8.79 ± 1.66 vs 20.86 ± 1.63% and seminal plasma arginase activity (0.12 ± 0.01 vs 0.22 ± 0.01 U/mL were significantly lower than in the non-stress situation, whereas seminal plasma NO (17.28 ± 0.56 vs 10.02 ± 0.49 µmol/L was higher compared to the non-stress period (P < 0.001 for all. During stress there was a negative correlation between NO concentration and sperm concentration, the percentage of rapid progressive motility and arginase activity (r = -0.622, P < 0.01; r = -0.425, P < 0.05 and r = -0.445, P < 0.05, respectively. These results indicate that psychological stress causes an increase of NO level and a decrease of arginase activity in the L-arginine-NO pathway. Furthermore, poor sperm quality may be due to excessive production of NO under psychological stress. In the light of these results, we suggest that the arginine-NO pathway, together with arginase and NO synthase, are involved in semen quality under stress conditions.

  17. Functional properties and stability of spray-dried pigments from Bordo grape (Vitis labrusca) winemaking pomace.

    Science.gov (United States)

    Souza, Volnei Brito de; Fujita, Alice; Thomazini, Marcelo; da Silva, Edson Roberto; Lucon, João Francisco; Genovese, Maria Inés; Favaro-Trindade, Carmen Sílvia

    2014-12-01

    The stability of anthocyanin and phenolic compounds, the antioxidant capacity, the antimicrobial activity and the capacity to inhibit arginase from Leishmania were evaluated in spray-dried powders from Bordo grape winemaking pomace extract. The pigments were produced using maltodextrin as the carrier agent at concentrations varying from 10% to 30% and air entrance temperatures varying from 130 to 170°C. A sample of freeze-dried extract without the carrier was also evaluated. The anthocyanins in the spray-dried samples showed good stability during storage, better than the freeze-dried and liquid extracts. The samples were capable of inhibiting the growth of Staphylococcus aureus and Listeria monocytogenes and showed high inhibitory capacity against the enzyme arginase from Leishmania. These results provide evidence that Bordo grapes from the winemaking process have the potential to be used as natural pigments with functional properties.

  18. Utilization of ornithine and arginine as specific precursors of clavulanic acid.

    Science.gov (United States)

    Romero, J; Liras, P; Martín, J F

    1986-01-01

    Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine. PMID:2877616

  19. Counter-regulatory paracrine actions of FGF-23 and 1,25(OH)2 D in macrophages.

    Science.gov (United States)

    Han, Xiaobin; Li, Linqiang; Yang, Jiancheng; King, Gwendalyn; Xiao, Zhousheng; Quarles, Leigh Darryl

    2016-01-01

    Mechanisms underlying the association between fibroblastic growth factor 23 (FGF-23) and inflammation are uncertain. We found that FGF-23 was markedly up-regulated in LPS/INF-γ-induced proinflammatory M1 macrophages and Hyp mouse-derived peritoneal macrophages, but not in IL-4-induced M2 anti-inflammatory macrophages. NF-КB and JAK/STAT1 pathways mediated the increased transcription of FGF-23 in response to M1 polarization. FGF-23 stimulated TNF-α, but not IL-6, expression in M0 macrophages and suppressed Arginase-1 expression in M2 macrophages through FGFR-mediated mechanisms. 1,25(OH)2 D stimulated Arginase-1 expression and inhibited FGF-23 stimulation of TNF-α. FGF-23 has proinflammatory paracrine functions and counter-regulatory actions to 1,25(OH)2 D on innate immune responses.

  20. Positive correlation of airway resistance and serum asymmetric dimethylarginine level in COPD patients with systemic markers of low-grade inflammation

    Science.gov (United States)

    Tajti, Gabor; Gesztelyi, Rudolf; Pak, Krisztian; Papp, Csaba; Keki, Sandor; Szilasi, Magdolna Emma; Mikaczo, Angela; Fodor, Andrea; Szilasi, Maria; Zsuga, Judit

    2017-01-01

    The major feature of COPD is a progressive airflow limitation caused by chronic airway inflammation and consequent airway remodeling. Modified arginase and nitric oxide synthase (NOS) pathways are presumed to contribute to the inflammation and fibrosis. Asymmetric dimethylarginine (ADMA) may shunt L-arginine from the NOS pathway to the arginase one by uncoupling and competitive inhibition of NOS and by enhancing arginase activity. To attest the interplay of these pathways, the relationship between ADMA and airflow limitation, described by airway resistance (Raw), was investigated in a cohort of COPD patients. Every COPD patient willing to give consent to participate (n=74) was included. Case history, laboratory parameters, serum arginine and ADMA, pulmonary function (whole-body plethysmography), and disease-specific quality of life (St George’s Respiratory Questionnaire) were determined. Multiple linear regression was used to identify independent determinants of Raw. The final multiple model was stratified based on symptom control. The log Raw showed significant positive correlation with log ADMA in the whole sample (Pearson’s correlation coefficient: 0.25, P=0.03). This association remained significant after adjusting for confounders in the whole data set (β: 0.42; confidence interval [CI]: 0.06, 0.77; P=0.022) and in the worse-controlled stratum (β: 0.84; CI: 0.25, 1.43; P=0.007). Percent predicted value of forced expiratory flow between 25% and 75% of forced vital capacity showed that significant negative, elevated C-reactive protein exhibited significant positive relationship with Raw in the final model. Positive correlation of Raw with ADMA in COPD patients showing evidence of a systemic low-grade inflammation implies that ADMA contributes to the progression of COPD, probably by shunting L-arginine from the NOS pathway to the arginase one.

  1. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats' Penile Tissues.

    Science.gov (United States)

    Oboh, Ganiyu; Ademiluyi, Adedayo O; Ademosun, Ayokunle O; Olasehinde, Tosin A; Oyeleye, Sunday I; Boligon, Aline A; Athayde, Margareth L

    2015-01-01

    This study was designed to determine the antioxidant properties and inhibitory effects of extract from Moringa oleifera leaves on angiotensin-I-converting enzyme (ACE) and arginase activities in vitro. The extract was prepared and phenolic (total phenols and flavonoid) contents, radical (nitric oxide (NO), hydroxyl (OH)) scavenging abilities, and Fe(2+)-chelating ability were assessed. Characterization of the phenolic constituents was done via high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. Furthermore, the effects of the extract on Fe(2+)-induced MDA production in rats' penile tissue homogenate as well as its action on ACE and arginase activities were also determined. The extract scavenged NO (∗) , OH (∗) , chelated Fe(2+), and inhibited MDA production in a dose-dependent pattern with IC50 values of 1.36, 0.52, and 0.38 mg/mL and 194.23 µg/mL, respectively. Gallic acid, chlorogenic acid, quercetin, and kaempferol were the most abundant phenolic compounds identified in the leaf extract. The extract also inhibited ACE and arginase activities in a dose-dependent pattern and their IC50 values were 303.03 and 159.59 µg/mL, respectively. The phenolic contents, inhibition of ACE, arginase, and Fe(2+)-induced MDA production, and radical (OH (∗) , NO (∗) ) scavenging and Fe(2+)-chelating abilities could be some of the possible mechanisms by which M. oleifera leaves could be used in the treatment and/or management of erectile dysfunction.

  2. Nitric Oxide-Related Biological Pathways in Patients with Major Depression.

    Directory of Open Access Journals (Sweden)

    Andreas Baranyi

    Full Text Available Major depression is a well-known risk factor for cardiovascular diseases and increased mortality following myocardial infarction. However, biomarkers of depression and increased cardiovascular risk are still missing. The aim of this prospective study was to evaluate, whether nitric-oxide (NO related factors for endothelial dysfunction, such as global arginine bioavailability, arginase activity, L-arginine/ADMA ratio and the arginine metabolites asymmetric dimethylarginine (ADMA and symmetric dimethylarginine (SDMA might be biomarkers for depression-induced cardiovascular risk.In 71 in-patients with major depression and 48 healthy controls the Global Arginine Bioavailability Ratio (GABR, arginase activity (arginine/ornithine ratio, the L-arginine/ADMA ratio, ADMA, and SDMA were determined by high-pressure liquid chromatography. Psychiatric and laboratory assessments were obtained at baseline at the time of in-patient admittance and at the time of hospital discharge.The ADMA concentrations in patients with major depression were significantly elevated and the SDMA concentrations were significantly decreased in comparison with the healthy controls. Even after a first improvement of depression, ADMA and SDMA levels remained nearly unchanged. In addition, after a first improvement of depression at the time of hospital discharge, a significant decrease in arginase activity, an increased L-arginine/ADMA ratio and a trend for increased global arginine bioavailability were observed.Our study results are evidence that in patients with major depression ADMA and SDMA might be biomarkers to indicate an increased cardiovascular threat due to depression-triggered NO reduction. GABR, the L-arginine/ADMA ratio and arginase activity might be indicators of therapy success and increased NO production after remission.

  3. Pathophysiology and Toxicokinetic Studies of Blue-Green Algae Intoxication in the Swine Model

    Science.gov (United States)

    1986-11-21

    Arginase Activity in Twelve Tissues &ad Serum- From- Market Height Swine-by a Direct Colorimetric Method o o o * # 145 Introduction .04...MATYRIALS RND METHODS Isotops Tritiated water was purchased from New England Nuclear Research Producto Company, Boston, MA, USA. The specific activity was 1...ARGINASI ACTI.VITY IN TWELVE TISSUES AND SERUM FROM MARKET WEIGHT SWINE BY A DIRECT COLORIMETRIC MEITHOD Randall A. Lovell, DVM Walter 1. Hoffmann, DYM

  4. Features of phospho- and amidohydrolases functioning in edaphotopes polluted by ore mill effluents

    Directory of Open Access Journals (Sweden)

    O. M. Artyushenko

    2006-02-01

    Full Text Available Influence of aerotechnogenic contamination of soils on activity of some hydrolytic enzymes of nitrogen and phosphorus cycles is examined. Biochemical mobilization of organophosphorous and nitrogen-bearing compounds in soils polluted by heavy metals is depressed to a variable extent. In descending order of sensitivity to the pollution, the studied enzymes ranked as follows: urease > alkaline phosphatase > arginase > АТPase > acid phosphatase > amidase.

  5. L-ornithine derived polyamines in cystic fibrosis airways.

    Directory of Open Access Journals (Sweden)

    Hartmut Grasemann

    Full Text Available Increased arginase activity contributes to airway nitric oxide (NO deficiency in cystic fibrosis (CF. Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.

  6. Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guo, Xue-Wu; Li, Yuan-Zi; Guo, Jian; Wang, Qing; Huang, Shi-Yong; Chen, Ye-Fu; Du, Li-Ping; Xiao, Dong-Guang

    2016-05-01

    Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

  7. Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

    Science.gov (United States)

    Kumar, Sunil; Saragadam, Tejaswani; Punekar, Narayan S

    2015-08-15

    Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

  8. Characterization of neutrophil subsets in healthy human pregnancies.

    Directory of Open Access Journals (Sweden)

    Aloysius Ssemaganda

    Full Text Available We have previously shown that in successful pregnancies increased arginase activity is a mechanism that contributes to the suppression of the maternal immune system. We identified the main type of arginase-expressing cells as a population of activated low-density granulocytes (LDGs in peripheral blood mononuclear cells and in term placentae. In the present study, we analyzed the phenotype of LDGs and compared it to the phenotype of normal density granulocytes (NDGs in maternal peripheral blood, placental biopsies and cord blood. Our data reveal that only LDGs but no NDGs could be detected in placental biopsies. Phenotypically, NDGs and LDGs from both maternal and cord blood expressed different levels of maturation, activation and degranulation markers. NDGs from the maternal and cord blood were phenotypically similar, while maternal, cord and placental LDGs showed different expression levels of CD66b. LDGs present in cord blood expressed higher levels of arginase compared to maternal and placental LDGs. In summary, our results show that in maternal and cord blood, two phenotypically different populations of neutrophils can be identified, whereas in term placentae, only activated neutrophils are present.

  9. Defence sugarcane glycoproteins disorganize microtubules and prevent nuclear polarization and germination of Sporisorium scitamineum teliospores.

    Science.gov (United States)

    Sánchez-Elordi, Elena; Baluška, František; Echevarría, Clara; Vicente, Carlos; Legaz, M Estrella

    2016-08-01

    Microtubules (MTs) are involved in the germination of Sporisorium scitamineum teliospores. Resistant varieties of sugar cane plants produce defence glycoproteins that prevent the infection of the plants by the filamentous fungi Sporisorium scitamineum. Here, we show that a fraction of these glycoproteins prevents the correct arrangement of MTs and causes nuclear fragmentation defects. As a result, nuclei cannot correctly migrate through the growing hyphae, causing germinative failure. Arginase activity contained in defence glycoproteins is already described for preventing fungal germination. Now, its enzymatically active form is presented as a link between the defensive capacity of glycoproteins and the MT disorganization in fungal cells. Active arginase is produced in healthy and resistant plants; conversely, it is not detected in the juice from susceptible varieties, which explains why MT depolarization, nuclear disorganization as well as germination of teliospores are not significantly affected by glycoproteins from non-resistant plants. Our results also suggest that susceptible plants try to increase their levels of arginase after detecting the presence of the pathogen. However, this signal comes "too late" and such defensive mechanism fails.

  10. Local tetrahydrobiopterin administration augments reflex cutaneous vasodilation through nitric oxide-dependent mechanisms in aged human skin.

    Science.gov (United States)

    Stanhewicz, Anna E; Bruning, Rebecca S; Smith, Caroline J; Kenney, W Larry; Holowatz, Lacy A

    2012-03-01

    Functional constitutive nitric oxide synthase (NOS) is required for full expression of reflex cutaneous vasodilation that is attenuated in aged skin. Both the essential cofactor tetrahydrobiopterin (BH(4)) and adequate substrate concentrations are necessary for the functional synthesis of nitric oxide (NO) through NOS, both of which are reduced in aged vasculature through increased oxidant stress and upregulated arginase, respectively. We hypothesized that acute local BH(4) administration or arginase inhibition would similarly augment reflex vasodilation in aged skin during passive whole body heat stress. Four intradermal microdialysis fibers were placed in the forearm skin of 11 young (22 ± 1 yr) and 11 older (73 ± 2 yr) men and women for local infusion of 1) lactated Ringer, 2) 10 mM BH(4), 3) 5 mM (S)-(2-boronoethyl)-l-cysteine + 5 mM N(ω)-hydroxy-nor-l-arginine to inhibit arginase, and 4) 20 mM N(G)-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS. Red cell flux was measured at each site by laser-Doppler flowmetry (LDF) as reflex vasodilation was induced. After a 1.0°C rise in oral temperature (T(or)), mean body temperature was clamped and 20 mM l-NAME was perfused at each site. Cutaneous vascular conductance was calculated (CVC = LDF/mean arterial pressure) and expressed as a percentage of maximum (%CVC(max); 28 mM sodium nitroprusside and local heat, 43°C). Vasodilation was attenuated at the control site of the older subjects compared with young beginning at a 0.3°C rise in T(or). BH(4) and arginase inhibition both increased vasodilation in older (BH(4): 55 ± 5%; arginase-inhibited: 47 ± 5% vs. control: 37 ± 3%, both P 0.05) at a 1°C rise in T(or). With a 1°C rise in T(or), local BH(4) increased NO-dependent vasodilation in the older (BH(4): 31.8 ± 2.4%CVC(max) vs. control: 11.7 ± 2.0%CVC(max), P vasodilation in aged human skin and that BH(4) NOS coupling mechanisms may be a potential therapeutic target for increasing skin blood flow during

  11. Activation of PPARγ in myeloid cells promotes lung cancer progression and metastasis.

    Directory of Open Access Journals (Sweden)

    Howard Li

    Full Text Available Activation of peroxisome proliferator-activated receptor-γ (PPARγ inhibits growth of cancer cells including non-small cell lung cancer (NSCLC. Clinically, use of thiazolidinediones, which are pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγ(flox/flox mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Mac(neg, or control PPARγ(flox/flox mice. In both models, mice receiving PPARγ-Mac(neg bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells.

  12. Urea production in long-term cultures of adult rat hepatocytes.

    Science.gov (United States)

    Sierra-Santoyo, A; López, M L; Hernández, A; Mendoza-Figueroa, T

    1994-04-01

    To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mm arginine or 0.4 mm ornithine and in cultures exposed to different concentrations of NH(4)Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mm NH(4)Cl showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.

  13. Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia.

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf; Aebischer, Toni

    2013-07-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH(4)(+) and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration.

  14. Giardia duodenalis Arginine Deiminase Modulates the Phenotype and Cytokine Secretion of Human Dendritic Cells by Depletion of Arginine and Formation of Ammonia

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf

    2013-01-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH4+ and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration. PMID:23589577

  15. Streptozotocin-induced diabetes in the rat is associated with changes in vaginal hemodynamics, morphology and biochemical markers

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    Munarriz Ricardo

    2006-05-01

    Full Text Available Abstract Background Diabetes is associated with declining sexual function in women. However, the effects of diabetes on genital tissue structure, innervation and function remains poorly characterized. In control and streptozotocin-treated female rats, we investigated the effects of diabetes on vaginal blood flow, tissue morphology, and expression of arginase I, endothelial nitric oxide synthase (eNOS and cGMP-dependent protein kinase (PKG, key enzymes that regulate smooth muscle relaxation. We further related these changes with estrogen receptor alpha (ERα and androgen receptor (AR expression. Results In addition to significantly elevated blood glucose levels, diabetic rats had decreased mean body weight, lower levels of plasma estradiol, and higher plasma testosterone concentration, compared to age-matched controls. Eight weeks after administration of buffer (control or 65 mg/kg of streptozotocin (diabetic, the vaginal blood flow response to pelvic nerve stimulation was significantly reduced in diabetic rats. Histological examination of vaginal tissue from diabetic animals showed reduced epithelial thickness and atrophy of the muscularis layer. Diabetic animals also had reduced vaginal levels of eNOS and arginase I, but elevated levels of PKG, as assessed by Western blot analyses. These alterations were accompanied by a reduction in both ERα and AR in nuclear extracts of vaginal tissue from diabetic animals. Conclusion In ovariectomized (estrogen deficient animals, previous reports from our lab and others have documented changes in blood flow, tissue structure, ERα, arginase I and eNOS that parallel those observed in diabetic rats. We hypothesize that diabetes may lead to multiple disruptions in sex steroid hormone synthesis, metabolism and action. These pathological events may cause dramatic changes in tissue structure and key enzymes that regulate cell growth and smooth muscle contractility, ultimately affecting the genital response during

  16. Arginine Metabolism in Myeloid Cells Shapes Innate and Adaptive Immunity

    Science.gov (United States)

    Rodriguez, Paulo C.; Ochoa, Augusto C.; Al-Khami, Amir A.

    2017-01-01

    Arginine metabolism has been a key catabolic and anabolic process throughout the evolution of the immune response. Accruing evidence indicates that arginine-catabolizing enzymes, mainly nitric oxide synthases and arginases, are closely integrated with the control of immune response under physiological and pathological conditions. Myeloid cells are major players that exploit the regulators of arginine metabolism to mediate diverse, although often opposing, immunological and functional consequences. In this article, we focus on the importance of arginine catabolism by myeloid cells in regulating innate and adaptive immunity. Revisiting this matter could result in novel therapeutic approaches by which the immunoregulatory nodes instructed by arginine metabolism can be targeted.

  17. Influence of black gram (Vigna mungo) trypsin inhibitory fraction on the hepatic protein catabolism in male albino mice.

    Science.gov (United States)

    Kamalakannan, V; Sathyamoorthy, A V; Motlag, D B

    1984-01-01

    The effect of black gram and black gram trypsin inhibitor on the protein catabolism of male albino mice has been investigated. Group 1 was given autoclaved black gram (control), Group II raw black gram and Group III the autoclaved black gram incorporated with 1% black gram trypsin inhibitor. Blood as well as urinary urea and creatine were found to be elevated in Groups II and III. Increased levels of arginase, ornithine transcarbamylase and transaminases were noted in Groups II and III. The results suggested an enhanced catabolism of proteins evoked by the native black gram trypsin inhibitor.

  18. Enhancement of Anti-Telomerase Immunity Against Prostate Cancer

    Science.gov (United States)

    2007-11-01

    cells in bone marrow of patients with stomach cancer. Adv Exp Med Biol 1998;451: 189-94. 7. Bronte V, Chappell DB, Apolloni E, et al. Unopposed...chain expression by L-arginine. J Biol Chem 2002;277: 21123-29. 13. Bronte V, Serafini P, De Santo C, et al. IL-4-induced arginase 1 suppresses...activity. Its dependence on IFN-gamma. J Immunol 1995;155: 15-26. 25. Mazzoni A, Bronte V, Visintin A, et al. Myeloid suppressor lines inhibit T

  19. [Enzyme levels and morphological picture of normal and cirrhotic rat livers following portal vein ligation and subcutaneous transposition of the spleen].

    Science.gov (United States)

    Zelder, O; Dorn, R; Bürcklein, H H; Bode, Ch; Bode, J C; Jerusalem, C R

    1975-01-01

    The effect of portal vein ligation after subcutaneous transposition of the spleen is investigated on enzyme-activities. and morphological pattern of the normal and cirrhotic rat-liver. The increase of glycolytic enzyme-activities and the decrease of enzyme-activities of oxidative metabolic pathways can be explained by adaptation on throttled blood supply of the liver. Significant decrease of arginase-activity (urea-cycle) can not be explained by reduced protein content of food (pair-fed-animals). Diminished substrate (ammonia)-level (NH3/t/hepatocytes) may be an explanation. Histological pattern of normal and cirrhotic rat liver is nearly unchanged after portal vein ligation.

  20. l-Arginine metabolism in cardiovascular and renal tissue from hyper- and hypothyroid rats

    Science.gov (United States)

    Moliz, Juan N; Quesada, Andrés; Montoro-Molina, Sebastian; Vargas-Tendero, Pablo; Osuna, Antonio; Wangensteen, Rosemary; Vargas, Félix

    2015-01-01

    This study assessed the effects of thyroid hormones on the enzymes involved in l-arginine metabolism and the metabolites generated by the different metabolic pathways. Compounds of l-arginine metabolism were measured in the kidney, heart, aorta, and liver of euthyroid, hyperthyroid, and hypothyroid rats after 6 weeks of treatment. Enzymes studied were NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]), arginases I and II, ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and l-arginine decarboxylase (ADC). Metabolites studied were l-arginine, l-citrulline, spermidine, spermine, and l-proline. Kidney heart and aorta levels of eNOS and iNOS were augmented and reduced (P < 0.05, for each tissue and enzyme) in hyper- and hypothyroid rats, respectively. Arginase I abundance in aorta, heart, and kidney was increased (P < 0.05, for each tissue) in hyperthyroid rats and was decreased in kidney and aorta of hypothyroid rats (P < 0.05, for each tissue). Arginase II was augmented in aorta and kidney (P < 0.05, for each tissue) of hyperthyroid rats and remained unchanged in all organs of hypothyroid rats. The substrate for these enzymes, l-arginine, was reduced (P < 0.05, for all tissues) in hyperthyroid rats. Levels of ODC and spermidine, its product, were increased and decreased (P < 0.05) in hyper- and hypothyroid rats, respectively, in all organs studied. OAT and proline levels were positively modulated by thyroid hormones in liver but not in the other tissues. ADC protein levels were positively modulated by thyroid hormones in all tissues. According to these findings, thyroid hormone treatment positively modulates different l-arginine metabolic pathways. The changes recorded in the abundance of eNOS, arginases I and II, and ADC protein in renal and cardiovascular tissues may play a role in the hemodynamic and renal manifestations observed in thyroid disorders. Furthermore, the changes in ODC and spermidine might

  1. Urea and its formation in coelacanth liver.

    Science.gov (United States)

    Brown, G W; Brown, S G

    1967-02-01

    Urea occurs in liver of the coelacanth Latimeria chalumnae to the extent of about 1.7 percent by weight. It was determined quantitatively by reaction with 1-phenyl-1,2-propanedione-2-oxime (Archibald reagent) and by measurement of ammonia released upon treatment with urease. Arginase and ornithine carbamoyltransferase, enzymes instrumental in the formation of urea in typical ureotelic vertebrates, occur in homogenates of coelacanth liver. Formed in part by the ornithine-urea cycle, urea may have an osmoregulatory function in the coelacanth as it has in elasmobranchs.

  2. Dual role of arginine metabolism in establishing pathogenesis.

    Science.gov (United States)

    Gogoi, Mayuri; Datey, Akshay; Wilson, Keith T; Chakravortty, Dipshikha

    2016-02-01

    Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.

  3. Biomarkers for detecting nitrogen deficiency during alcoholic fermentation in different commercial wine yeast strains.

    Science.gov (United States)

    Gutiérrez, Alicia; Chiva, Rosana; Beltran, Gemma; Mas, Albert; Guillamon, José Manuel

    2013-05-01

    Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Several putative biomarkers were tested in order to analyze their appropriateness to detect nitrogen stress in the yeast. To this aim, four commercial wine strains (PDM, ARM, RVA and TTA) were grown in a synthetic grape must with different nitrogen concentrations. Trehalose accumulation, arginase activity and the expression of eleven genes were tested in these wine strains, known to have different nitrogen requirements. The overall response of the four strains was similar, with differences in response intensity (PDM and RVA with higher intensity) and response time (which was also related with nitrogen consumption time). Trehalose response was mostly related to entry into the stationary phase, whereas arginase activity was responsive to nitrogen depletion, although its measurement is too complicated to be used for routine monitoring during winemaking. The expression of the genes DAL4, DAL5, DUR3 and GAP1 was clearly related to nitrogen depletion and thus, GAP1 and DAL4 were selected as markers of nitrogen deficiency. In order to adapt expression analysis to winemaking conditions, the original strains were transformed into reporter strains based on the expression of green fluorescent protein (GFP) under control of the promoters for GAP1 and DAL4. The transformants had a similar fermentative capacity to the parental strains and were able to detect alterations in yeast physiological status due to nitrogen limitations.

  4. Hepatoprotective, antioxidant, and ameliorative effects of ginger (Zingiber officinale Roscoe) and vitamin E in acetaminophen treated rats.

    Science.gov (United States)

    Abdel-Azeem, Amal S; Hegazy, Amany M; Ibrahim, Khadiga S; Farrag, Abdel-Razik H; El-Sayed, Eman M

    2013-09-01

    Ginger is a remedy known to possess a number of pharmacological properties. This study investigated efficacy of ginger pretreatment in alleviating acetaminophen-induced acute hepatotoxicity in rats. Rats were divided into six groups; negative control, acetaminophen (APAP) (600 mg/kg single intraperitoneal injection); vitamin E (75 mg/kg), ginger (100 mg/kg), vitamin E + APAP, and ginger + APAP. Administration of APAP elicited significant liver injury that was manifested by remarkable increase in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), arginase activities, and total bilirubin concentration. Meanwhile, APAP significantly decreased plasma total proteins and albumin levels. APAP administration resulted in substantial increase in each of plasma triacylglycerols (TAGs), malondialdhyde (MDA) levels, and total antioxidant capacity (TAC). However, ginger or vitamin E treatment prior to APAP showed significant hepatoprotective effect by lowering the hepatic marker enzymes (AST, ALT, ALP, and arginase) and total bilirubin in plasma. In addition, they remarkably ameliorated the APAP-induced oxidative stress by inhibiting lipid peroxidation (MDA). Pretreatment by ginger or vitamin E significantly restored TAGs, and total protein levels. Histopathological examination of APAP treated rats showed alterations in normal hepatic histoarchitecture, with necrosis and vacuolization of cells. These alterations were substantially decreased by ginger or vitamin E. Our results demonstrated that ginger can prevent hepatic injuries, alleviating oxidative stress in a manner comparable to that of vitamin E. Combination therapy of ginger and APAP is recommended especially in cases with hepatic disorders or when high doses of APAP are required.

  5. [Blood monocytic L-arginine metabolic changes in diabetic foot syndrome].

    Science.gov (United States)

    Barinov, E F; Sulaeva, O N; Barinova, M E

    2010-05-01

    An inhibition test was used to study mechanisms responsible for L-arginine metabolic disturbances in the blood monocytes of patients with diabetic foot syndrome (DFS). It showed enhanced baseline iNOS activity and inhibition of the arginase pathway with lower nitrite production in response to the administration of lipopolysaccharide in the monocytes of patients with DFS. Impaired L-arginine metabolism was related to the higher activities of protein kinase C (PKC), phosphodiesterase (PDE), and 5-lipoxygenase (5-LO) along with decreased cyclooxygenase activity and drastic protein kinase A (PKA) inhibition. Within the first week, no changes in the wound process were associated with persistent metabolic disturbances of arachidonic acid and serine-threonine kinases with the higher sensitivity of AT1 receptors. In patients with DFS, the condition for wound process termination was decreased baseline iNOS activity and enhanced arginase-1 activity during PKA stimulation with the lower activity of 5-LO, PDE, and PKS. However, impaired mechanisms in the regulation of monocytic L-arginine metabolism persisted even a month later, which predetermines skin remodeling disturbance and the likelihood of recurrent DFS

  6. Biochemical and morphological effects of polyamine biosynthesis inhibitors on Trichophyton and Microsporum.

    Science.gov (United States)

    Gruhn, C M; Boyle, S M

    1991-01-01

    The minimum inhibitory concentrations (MICs) of three known irreversible inhibitors of polyamine synthesis, alpha-difluoromethylornithine (DFMO) and monofluoromethyldehydroornithine methylester (MFMOme), inhibitors of ornithine decarboxylase (ODC) and alpha-difluoromethylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC), were determined for 10 species of dermatophytic fungi. Trichophyton species were generally more sensitive to these inhibitors than Microsporum species. Both genera produced arginase, and treatment of members of either genus with DFMO or DFMA resulted in an inhibition of ODC activity and a depletion of cellular polyamines. However, conversion of labelled DFMA to DFMO, either in vivo or in vitro, could not be demonstrated in spite of both genera producing arginase. The ultrastructure of cells cultured in the presence of either DFMO or DFMA was similar, and revealed disruption of calcium metabolism, an increase in mitochondrial number and alterations to membrane systems. DFMA and DFMO also inhibited sporulation in Microsporum gypseum. Our findings indicate that DFMO limits the growth of dermatophytes by direct inhibition of ODC and lowering of cellular polyamine levels; in contrast, DFMA inhibits polyamine synthesis in an unspecified manner as ADC activity was undetected.

  7. Inhibition of polyamine biosynthesis in Crithidia fasciculata by D,L-alpha-difluoromethylornithine and D,L-alpha-difluoromethylarginine.

    Science.gov (United States)

    Hunter, K J; Strobos, C A; Fairlamb, A H

    1991-05-01

    Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of D,L-alpha-difluoromethylornithine (DFMO) and D,L-alpha-difluoromethylarginine (DFMA) on growth and polyamine synthesis. In a defined, polyamine-free medium growth was markedly inhibited by DFMO (94% at 50 mM; IC50 = 37 mM) and to a lesser extent by DFMA (65% at 50 mM). Addition of putrescine, but not agmatine, reverses inhibition of growth, suggesting that the site of inhibition is ornithine decarboxylase (ODC). Consistent with this conclusion, DFMO or DFMA results in a complete loss of putrescine and significant reductions in intracellular spermidine, glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione). In addition, significant concentrations of DFMO (0.8 mM) were present in DFMA-treated cells. However, in contrast to other organisms, conversion of DFMA to DFMO is probably not catalysed by arginase. Substantial ornithine decarboxylase activity (63.1 pmol min-1 mg-1; ODC) was observed in control cells, sufficient to account for polyamine synthesis during growth. In addition, a trace arginine decarboxylase (ADC) activity (1.19 pmol min-1 mg-1) was found. Evidence is presented showing that the apparent ADC activity is actually due to the concerted action of arginase (1.5 nmol min-1 mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC.

  8. The predominance of alternatively activated macrophages following challenge with cell wall peptide-polysaccharide after prior infection with Sporothrix schenckii.

    Science.gov (United States)

    Alegranci, Pamela; de Abreu Ribeiro, Livia Carolina; Ferreira, Lucas Souza; Negrini, Thais de Cássia; Maia, Danielle Cardoso Geraldo; Tansini, Aline; Gonçalves, Amanda Costa; Placeres, Marisa Campos Polesi; Carlos, Iracilda Zeppone

    2013-08-01

    Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into "classic" or "alternatively" activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-β, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-β production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.

  9. NITRIC OXIDE (NO, CITRULLINE - NO CYCLE ENZYMES, GLUTAMINE SYNTHETASE AND OXIDATIVE STRESS IN ANOXIA (HYPOBARIC HYPOXIA AND REPERFUSION IN RAT BRAIN

    Directory of Open Access Journals (Sweden)

    M. Swamy, Mohd Jamsani Mat Salleh, K. N .S. Sirajudeen, Wan Roslina Wan Yusof, G. Chandran

    2010-01-01

    Full Text Available Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia and reperfusion (reoxygenation, the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite, thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex, cerebellum and brain stem of rats subjected to anoxia and reperfusion. The results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase. The increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia, making nitric oxide production more effective and contributing to its toxic effects. The decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia. The increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion. The increased arginase and sustained decrease of GS activity in reperfusion group likely to be protective.

  10. Role of dietary fish oil on nitric oxide synthase activity and oxidative status in mice red blood cells.

    Science.gov (United States)

    Martins, Marcela A; Moss, Monique B; Mendes, Iara K S; Águila, Márcia B; Mandarim-de-Lacerda, Carlos Alberto; Brunini, Tatiana M C; Mendes-Ribeiro, Antônio Cláudio

    2014-12-01

    The consumption of n-3 polyunsaturated fatty acids (PUFAs) derived from fish oil concomitant with a reduced intake of saturated fats is associated with cardiovascular benefits, which may result from the participation of nitric oxide (NO). In contrast, PUFAs are vulnerable to peroxidation, which could affect the oxidative stability of the cell and reduce NO bioavailability. Therefore, we investigated the effects of high fat diets with increasing amounts of fish oil (0-40% of energy) in place of lard on the l-arginine-NO pathway, the arginase pathway and oxidative status in mice red blood cells (RBC). We found that l-arginine transport, as well as NO synthase (NOS) expression and activity, was enhanced by the highest doses of fish oil (30 and 40%). In contrast, diets rich in lard led to NOS expression and activity impairment. Arginase expression was not significantly affected by any of the dietary regimens. No significant difference in protein and lipid oxidative markers was observed among any of the fish-oil fed mice; only lard feeding induced protein damage in addition to a decreased superoxide dismutase activity. These data suggest that a substantial dose of fish oil, but not low doses, activates the RBC l-arginine-NO pathway without resulting in oxidative damage.

  11. Oxidized LDL Induces Alternative Macrophage Phenotype through Activation of CD36 and PAFR

    Directory of Open Access Journals (Sweden)

    Francisco J. Rios

    2013-01-01

    Full Text Available OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR.

  12. Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner.

    Science.gov (United States)

    Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M; Wiltrout, Robert H; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A; Manns, Michael P; Wang, Ena; Marincola, Francesco M; Korangy, Firouzeh; Greten, Tim F

    2015-04-01

    Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.

  13. [Urea formation in the after operational liver].

    Science.gov (United States)

    Savilov, P N

    2016-01-01

    The effect of resection of the left lobe of the liver (LR, 15-20% og the organ weight) on hepatic urea formation was investigated in 84 albino rats. The objects of study were the surgery left (LLP), inoperable middle (MLP) lobe of the liver, blood (aorta, v. hepatica, v. porta) and choledochal bile. They studied the urea content. Arginase activity was examined in liver homogenate. On the day 3 and day 7 after resection reduced arginase activity was detected. LR caused a decrease of urea in v. hepatica, but increased urea content in the arterial blood and v. porta. Increase in bile urea on day 7 it was replaced by a decrease observed on day 14 of the postsurgery period. The concentration of urea in the liver on the 3rd day after LR was below the norm, and on the 7th and 14th day was within it. The results indicate a violation of urea operated by hepatocytes of the liver and extrahepatic activation mechanisms of the formation of urea.

  14. Supplemental carvacrol can reduce the severity of inflammation by influencing the production of mediators of inflammation.

    Science.gov (United States)

    Kara, Mehmet; Uslu, Sema; Demirci, Fatih; Temel, Halide Edip; Baydemir, Canan

    2015-01-01

    Carvacrol (CVC) is a monoterpenic phenol, which is present in the essential oil of various plants. It has been widely used both as antibacterial feed additive and food preservative. Therefore, our objective was to evaluate the prophylactic effects of carvacrol on inflammatory mediators of sepsis. Serum tumor necrosis factor alpha and interleukin 6 levels as proinflammatory markers were evaluated using an enzyme-linked immunosorbent assay technique. Malondialdehyde (MDA) was determined in the sample by using thiobarbituric acid test. Nitric oxide (NO) levels and arginase activity and also all measurements were evaluated after 24 h from lipopolysaccharide (LPS) injections done (1 mg/kg i.p.). All carvacrol doses (20, 40, and 80 mg/kg) were given by intra gastric gavage during six days before LPS injection (7th day). Proinflammatory cytokines, MDA, NO levels, and arginase activity were decreased by carvacrol according to the carvacrol doses. These results indicate that carvacrol may have a potent anti-inflammatory and antioxidant effects in a dose-dependent manner. Subchronic use of CVC can be assisted to pre-treat of sepsis as a prophylactic.

  15. Circulating myeloid-derived suppressor cells in patients with pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Xu; Jun Hu; Min Wang; Feng Peng; Rui Tian; Xing-Jun Guo; Yu Xie; Ren-Yi Qin

    2016-01-01

    BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cell types that suppress T-cell responses in cancer patients and animal models, some MDSC subpopula-tions are increased in patients with pancreatic cancer. The present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients. METHODS: Myeloid cells from whole blood were collected from 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, and 47 healthy controls. Four pancreatic cancer cell lines were co-culturedwithnormalperipheralbloodmononuclearcells(PBMCs) to test the effect of tumor cells on the conversion of PBMCs to MDSCs. Levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and arginase activity in the plasma of cancer patients were analyzed by enzyme-linked immunosorbent assay. RESULTS: CD14+/CD11b+/HLA-DR- MDSCs were increased in patients with pancreatic or bile duct cancer compared with those in healthy controls, and this increase was correlated with clinical cancer stage. Pancreatic cancer cell lines induced PBMCs to MDSCs in a dose-dependent manner. GM-CSF and arginase activity levels were significantly increased in the se-rum of patients with pancreatic cancer. CONCLUSIONS: MDSCsweretumorrelated:tumorcellsinduced PBMCs to MDSCs in a dose-dependent manner and circulating CD14+/CD11b+/HLA-DR- MDSCs in pancreatic cancer patients were positively correlated with tumor burden. MDSCs might be useful markers for pancreatic cancer detection and progression.

  16. Cruzipain, a major Trypanosoma cruzi antigen, conditions the host immune response in favor of parasite.

    Science.gov (United States)

    Giordanengo, Laura; Guiñazú, Natalia; Stempin, Cinthia; Fretes, Ricardo; Cerbán, Fabio; Gea, Susana

    2002-04-01

    We recently demonstrated that humoral immune response to cruzipain, a major antigen of Trypanosoma cruzi parasite, is implicated in the pathogenesis of experimental Chagas' disease. In the present study, the spleen cell phenotype and the cytokine profile induced by cruzipain in immunized mice were analyzed. The results showed that cruzipain increases the number of spleen cells with large size and granularity. Splenocyte populations with CD19(+), Mac-1(+), Gr-1(+) and CD11c(+) positive surface markers significantly increased in immune mice compared to controls ones. Histological study revealed the presence of high number of megacariocyte and granulocyte-macrophage progenitors, indicating extramedullary hemopoiesis in spleens of immune mice. The finding of high levels of IL-4, IL5 and IL-10 and low levels of IFN-gamma and IL-12 in supernatants of immune cells stimulated with cruzipain indicates a preferential activation of T2 type cells in immune animals. To investigate the role of innate immunity cells, the classical and alternative metabolic pathways of spleen macrophages from immune mice stimulated by cruzipain were also studied. The results showed an increase of urea associated with a decrease of nitrite levels, suggesting that cruzipain up-regulates the arginase way. Therefore, cruzipain leads to T2 type cytokine profile which may enhance the arginase via in the macrophages promoting a susceptible mechanism to infection. Thus, we postulate that during T. cruzi infection, cruzipain could be used by the parasite to spread inside the host.

  17. Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen.

    Science.gov (United States)

    Stempin, Cinthia; Giordanengo, Laura; Gea, Susana; Cerbán, Fabio

    2002-10-01

    We studied the macrophage (Mo) activation pathways through Mo interaction with immunogenic Trypanosoma cruzi antigens as cruzipain (Cz) and R13. J774 cells, peritoneal and spleen Mo from normal mice, were used. Although Mo classic activation was observed in the presence of lipopolysaccharide, evaluated through nitric oxide (NO) and interleukin (IL)-12 production, Cz and R13 did not activate Mo in this way. To study the alternative pathway, we examined the arginase activity in Mo cultured with Cz. An increase of arginase activity was detected in all Mo sources assayed. An increase of IL-10 and transforming growth factor-beta in culture supernatants from Mo stimulated with Cz was observed. The study of expression of B7.1 and B7.2 in spleen Mo revealed that Cz induces preferential expression of B7.2. In vitro studies revealed that Cz stimulated J774 cells and then, infected with trypomastigotes of T. cruzi, developed a higher number of intracellular parasites than unstimulated infected Mo. Thus, Cz favors the perpetuation of T. cruzi infection. In addition, a down-regulation of inducible NO synthase was observed in J774 cells stimulated with Cz. These results suggest that Cz interaction with Mo could modulate the immune response generated against T. cruzi through the induction of a preferential metabolic pathway in Mo.

  18. Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

    Science.gov (United States)

    Motrán, Claudia Cristina; Díaz, Fernando López; Gruppi, Adriana; Slavin, Daniela; Chatton, Bruno; Bocco, José Luis

    2002-09-01

    It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

  19. Topographical modulation of macrophage phenotype by shrink-film multi-scale wrinkles.

    Science.gov (United States)

    Wang, Tingting; Luu, Thuy U; Chen, Aaron; Khine, Michelle; Liu, Wendy F

    2016-06-24

    The host immune response to foreign materials is a major hurdle for implanted medical devices. To control this response, modulation of macrophage behavior has emerged as a promising strategy, given their prominent role in inflammation and wound healing. Towards this goal, we explore the effect of biomimetic multi-scale wrinkles on macrophage adhesion and expression of phenotype markers. We find that macrophages elongate along the direction of the uniaxial wrinkles made from shape memory polymers, and express more arginase-1 and IL-10, and less TNF-α, suggesting polarization towards an alternatively activated, anti-inflammatory phenotype. Materials were further implanted in the subcutaneous space of mice and tissue surrounding the material evaluated by histology and immunohistochemistry. We found that material surface topography altered the distribution of collagen deposition in the adjacent tissue, with denser collagen tissue observed near flat materials when compared to wrinkled materials. Furthermore, cells surrounding wrinkled materials exhibited higher arginase-1 expression. Together these data suggest that wrinkled material surfaces promote macrophage alternative activation, and may influence the foreign body response to implants.

  20. Effects of temperature, food deprivation and salinity on growth, RNA/DNA ratio and certain enzyme activities in rainbow trout (Salmo gairdneri Richardson).

    Science.gov (United States)

    Jürss, K; Bittorf, T; Vökler, T; Wacke, R

    1987-01-01

    1. The connection between feeding regime (food deprivation and restricted diet) and thermal acclimation (1-2, 6, 11 and 16 degrees C) was studied in rainbow trout held in diluted seawater (20% S). 2. At 1 degree C, food deprivation effects on all parameters are slight, and on RNA and certain enzymes they are masked by thermal acclimation effects. 3. At a salinity of 20% rainbow trout on a restricted diet and held at 11 degrees C have the highest growth rate. 4. Owing to increasing RNA levels, the RNA/DNA quotient is significantly higher than normal in rainbow trout held at 1 degree C although the fishes do not grow at this temperature. 5. Temperature and feeding both affect the enzymes we studied (liver: G1DH, AspT, arginase, G6PDH, and 6PGDH; kidney: G1DH, AspT, arginase, and Na/K-ATPase; white muscle: AspT and A1T; gill: Na/K-ATPase) differently. Interactions between these two factors also occur in some cases.

  1. Development and optimization of a novel conductometric bi-enzyme biosensor for L-arginine determination.

    Science.gov (United States)

    Saiapina, O Y; Dzyadevych, S V; Jaffrezic-Renault, N; Soldatkin, O P

    2012-04-15

    A highly sensitive conductometric biosensor for l-arginine determination was developed by exploiting the unique biorecognition capacities of two enzymes of urea cycle - arginase (E.C. 3.5.3.1) and urease (E.C. 3.5.1.5). The enzymes were co-immobilized in a single bioselective membrane on the working sensor, while a lysine rich bovine serum albumin (BSA) membrane was immobilized on the reference sensor, allowing differential measurements. The optimum percentage ratio of arginase and urease within the bioselective membrane was determined when the biosensor sensitivity to l-arginine and urea was optimum. Analytical characteristics of the conductometric biosensor for l-arginine determination were compared for two types of enzyme immobilization (cross-linking with glutaraldehyde (GA) and entrapment in the polymeric membrane). The optimum features in terms of the sensitivity, the linear range, and the detection limit (4.2 μS/mM, 0.01-4mM, and 5.0 × 10(-7)M, respectively) were found for l-arginine biosensor based on enzyme cross-linking with GA. A quantitative determination of l-arginine in the real sample (a drinkable solution "Arginine Veyron") gave a satisfactory result compared to the data provided by the producer (a relative error was 4.6%). The developed biosensor showed high operational and storage stability.

  2. The absence of MyD88 has no effect on the induction of alternatively activated macrophage during Fasciola hepatica infection

    Directory of Open Access Journals (Sweden)

    Luo HongLin

    2011-11-01

    Full Text Available Abstract Background Alternatively activated macrophages (AAMϕ play important roles in allergies and responses to parasitic infections. However, whether signaling through toll-like receptors (TLRs plays any role in AAMϕ induction when young Fasciola hepatica penetrates the liver capsule and migrates through the liver tissue is still unclear. Results The data show that the lack of myeloid differentiation factor 88 (MyD88 has no effect on the AAMϕ derived from the bone marrow (BMMϕ in vitro and does not impair the mRNA expression of arginase-1, resistin-like molecule (RELMα, and Ym1 in BMMϕs. The Th2 cytokine production bias in splenocytes was not significantly altered in F. hepatica-infected mice in the absence of MyD88 in vitro and in the pleural cavity lavage in vivo. In addition, MyD88-deficiency has no effect on the arginase production of the F. hepatica elicited macrophages (Fe Mϕs, production of RELMα and Ym1 proteins and mRNA expression of Ym1 and RELMα of macrophages in the peritoneal cavity 6 weeks post F. hepatica infection. Conclusions The absence of MyD88 has no effect on presence of AAMϕ 6 weeks post F. hepatica infection.

  3. Protective effects of sodium orthovanadate in diabetic reticulocytes and ageing red blood cells of Wistar rats

    Indian Academy of Sciences (India)

    Bihari L Gupta; Anju Preet; Najma Z Baquer

    2004-03-01

    The reticulocytes and the ageing red blood cells (RBCs) namely young (Y), middle-aged (M) and old RBCs (O) of female Wistar rats from different groups such as control animals (C), controls treated with vanadate (C + V), alloxan-induced diabetic (D), diabetic-treated with insulin (D + I) and vanadate (D + V), were fractionated on a percoll/BSA gradient. The following enzymes were measured – hexokinase (HK), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione-s-transferase (GST), alanine aminotransferase (AlaAT), aspartate aminotransferase (AsAT) and arginase in the hemolysates of all the RBCs fractions. Decreases in the activity of HK and AsAT by about 70%, arginase and GSH-Px by 30% in old RBCs were observed in comparison to reticulocytes of control animals. Increases in the activity of GSSG-R by 86%, AlaAT by more than 400% and GST by 70% were observed in old RBCs in comparison to reticulocytes of control animals. Alloxan diabetic animals showed a further decrease in the activities of HK in Y RBCs by 37%, M RBCs by 39% and O RBCs by 32%, GSH-Px activity in Y RBCs by 13%, M RBCs by 20% and O RBCs by 33% and GST activity in Y RBCs by 14%, M RBCs by 42% and O RBCs by 60% in comparison to their corresponding cells of control animals. An increase in the activity of all the enzymes studied was also observed in reticulocytes of diabetic animals in comparison to reticulocytes of controls. The GSSG-R activity was found to be increased in Y RBCs by 49%, M RBCs by 67% and O RBCs by 64% as compared to the corresponding age-matched cells of control animals. The activity of arginase also decreased in Y RBCs by about10%, M RBCs by 20% and O RBCs by 30% in comparison to the age-matched cells of control animals. A decrease in the activity of AsAT in Y and M RBCs by 30%, and O RBCs by 25% was observed in diabetic animals in comparison to the age-matched cells of control animals. The activity of AlaAT was found to be decreased by more than 10% in Y and M

  4. Electroacupuncture at the ST36 acupoint increases interleukin-4 responsiveness in macrophages, generation of alternatively activated macrophages and susceptibility to Leishmania major infection

    Directory of Open Access Journals (Sweden)

    Aguiar Danillo N

    2012-07-01

    Full Text Available Abstract Background Electroacupuncture (EA has been used to treat inflammatory diseases. Alternatively activated macrophages (AAMo stimulated by cytokines such as interleukin (IL-4, IL-10 and IL-13 are anti-inflammatory and mildly microbicidal. This study aimed to evaluate whether EA at the Zusanli acupoint (ST36 would change the profile of healthy murine macrophages, particularly the generation of AAMo and susceptibility to Leishmania major infection. Methods BALB/c mice were treated with EA (15/30 Hz at the ST36 acupoint for 20 min/d for 5 d. After the final EA session, the mice were euthanized and their peritoneal cells were harvested and counted for determination of arginase activity, nitric oxide (NO production and microbicidal activity after culture in the presence or absence of IL-4, interferon-γ (IFNγ or lipopolysaccharide (LPS or both IFNγ and LPS. Twelve mice were infected with L. major promastigotes into the footpads after the final EA session and the infection course was monitored. Results Peritoneal cells freshly obtained from EA-treated mice had similar arginase and microbicidal activities to cells from sham-treated mice. After culture with IL-4, cells from EA-treated mice exhibited significant increases in the arginase activity (sham: 58 ± 11.3 vs. EA: 80.7 ± 4.6%, P = 0.025 and number of parasites/infected cell (sham: 2.5 ± 0.4 vs. EA: 4.3 ± 0.8 cells, P = 0.007. The NO production was lower in cells from EA-treated mice cultured in the presence of a combination of IFNγ and LPS (sham: 31.6 ± 6.5 vs. EA: 22.3 ± 2.1 μM, P = 0.025. The lesion size in mice infected with L. major promastigotes was larger in EA-treated mice (sham: 3.26 ± 0.29 vs. EA: 2.23 ± 0.4 mm, P = 0.039. Conclusion EA at the ST36 acupoint increases IL-4 responsiveness in macrophages, Generation of AAMo and susceptibility to L. major infection

  5. Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L.

    Science.gov (United States)

    Desmaison, A M; Tixier, M

    1986-06-01

    During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, gamma-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.

  6. Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

    Science.gov (United States)

    Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

    2014-01-01

    The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8.

  7. The nutritional management of urea cycle disorders.

    Science.gov (United States)

    Leonard, J V

    2001-01-01

    Diet is one of the mainstays of the treatment of patients with urea cycle disorders. The protein intake should be adjusted to take account of the inborn error and its severity and the patient's age, growth rate, and individual preferences. Currently, the widely used standards for protein intake are probably more generous than necessary, particularly for those with the more severe variants. Most patients, except those with arginase deficiency, will need supplements of arginine, but the value of other supplements including citrate and carnitine is unclear. Any patient on a low-protein diet should be monitored clinically and with appropriate laboratory tests. All should have an emergency (crisis) regimen to prevent decompensation during periods of metabolic stress.

  8. TNF Receptor-2 Facilitates an Immunosuppressive Microenvironment in the Liver to Promote the Colonization and Growth of Hepatic Metastases

    DEFF Research Database (Denmark)

    Ham, Boram; Wang, Ni; D'Costa, Zarina

    2015-01-01

    . In this study, we explored how TNF signaling influences the efficiency of liver metastasis by colon and lung carcinoma in mice that are genetically deficient for the TNF receptor TNFR2. We found a marked reduction in liver metastases that correlated with a greatly reduced accumulation at metastatic sites of CD......11b(+)GR-1(+) myeloid cells with enhanced arginase activity, identified as myeloid-derived suppressor cells (MDSC). Reduced infiltration of MDSC coincided with a reduction in the number of CD4(+)FoxP3(+) T regulatory cells in the tumors. Reconstitution of TNFR2-deficient mice with normal bone marrow......, or adoptive transfer of TNFR2-expressing MDSC into these mice, was sufficient to restore liver metastasis to levels in wild-type mice. Conversely, treatment with TNFR2 antisense oligodeoxynucleotides reduced liver metastasis in wild-type mice. Clinically, immunohistochemical analysis of liver metastases from...

  9. Lentiviral-mediated administration of IL-25 in the CNS induces alternative activation of microglia

    DEFF Research Database (Denmark)

    Maiorino, C; Khorooshi, R; Ruffini, F

    2013-01-01

    Interleukin-25 (IL-25) is the only anti-inflammatory cytokine of the IL-17 family, and it has been shown to be efficacious in inhibiting neuroinflammation. Known for its effects on cells of the adaptive immune system, it has been more recently described to be effective also on cells of the innate...... immune system, namely macrophages. We used a lentiviral-mediated gene therapy approach to deliver IL-25 to the central nervous system (CNS) in two mouse models of neuroinflammation, entorhinal cortex lesion and experimental autoimmune encephalomyelitis. In both, we found that IL-25 gene therapy was able...... to modulate CNS myeloid cells, either infiltrating macrophages or resident microglia, towards an anti-inflammatory, tissue-protective phenotype, as testified by the increase in markers such as Arginase-1 (Arg1), Mannose receptor 1 (CD206) and Chitinase 3-like 3 (Ym1). As a consequence, neuroinflammation...

  10. Immunology and immunopathology of African trypanosomiasis

    Directory of Open Access Journals (Sweden)

    Philippe Vincendeau

    2006-12-01

    Full Text Available Major modifications of immune system have been observed in African trypanosomiasis. These immune reactions do not lead to protection and are also involved in immunopathology disorders. The major surface component (variable surface glycoprotein,VSG is associated with escape to immune reactions, cytokine network dysfunctions and autoantibody production. Most of our knowledge result from experimental trypanosomiasis. Innate resistance elements have been characterised. In infected mice, VSG preferentially stimulates a Th 1-cell subset. A response of gd and CD8 T cells to trypanosome antigens was observed in trypanotolerant cattle. An increase in CD5 B cells, responsible for most serum IgM and production of autoantibodies has been noted in infected cattle. Macrophages play important roles in trypanosomiasis, in synergy with antibodies (phagocytosis and by secreting various molecules (radicals, cytokines, prostaglandins,.... Trypanosomes are highly sensitive to TNF-alpha, reactive oxygen and nitrogen intermediates. TNF-alpha is also involved in cachexia. IFN-gamma acts as a parasite growth factor. These various elements contribute to immunosuppression. Trypanosomes have learnt to use immune mechanisms to its own profit. Recent data show the importance of alternative macrophage activation, including arginase induction. L-ornithine produced by host arginase is essential to parasite growth. All these data reflect the deep insight into the immune system realised by trypanosomes and might suggest interference therapeutic approaches.Modificações importantes no sistema imune são observadas na tripanosomíase Africana. Essas reações imunológicas não protegem e estão envolvidas em distúrbios imunopatológicos. O principal componente de superfície (glicoproteína variante de superfície, VSG está associado à evasão das respostas imune, às disfunções da rede de citocinas e à produção de autoanticorpos. Muitos de nossos conhecimentos resultam

  11. Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning

    DEFF Research Database (Denmark)

    Tygstrup, N; Jensen, S A; Krog, B;

    1996-01-01

    AIM: In order to study the short-term effect of moderate and severe reduction of liver function by acetaminophen poisoning of different severity on gene expression for liver-specific functions, rats were given 3.75 and 7.5 g per kg body weight acetaminophen intragastrically. The lower dose...... involved in metabolic liver functions, i.e. ureagenesis, gluconeogenesis, and drug metabolism, for acute phase proteins, "house-keeping" proteins, and for proteins related to liver regeneration. Results were expressed as per cent of the level in similarly fasted, untreated rats of the same stock RESULTS...... towards the end of the experiment. The greatest differences were seen for mRNA of arginase, beta-fibrinogen, alpha 1-acid glycoprotein, alpha-tubulin, histone 3, TGF beta, and cyclin d, i.e. proteins associated with acute phase response and liver cell replication and maintenance. CONCLUSIONS...

  12. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    Science.gov (United States)

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  13. CHANGES IN SERUM ENZYMES LEVELS ASSOCIATED WITH LIVER FUNCTIONS IN STRESSED MARWARI GOAT

    Directory of Open Access Journals (Sweden)

    Kataria N.

    2011-03-01

    Full Text Available Serum enzyme levels were determined in goats of Marwari breed belonging to farmers’ stock of arid tract of Rajasthan state, India. The animals were grouped into healthy and stressed comprising of gastrointestinal parasiticised, pneumonia affected, and drought affected. The serum enzymes determined were sorbitol dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase, gamma-glutamayl transferase, 5’nucleotidase, glucose-6-phosphatase, arginase, and aldolase. In stressed group the mean values of all the enzymes increased significantly (p≤0.05 as compared to respective healthy mean value. All the enzymes showed highest values in the gastrointestinal parasiticised animals and least values in the animals having pneumonia. In gastrointestinal parasiticised animals maximum change was observed in G-6-Pase activity and minimum change was observed in malate dehydrogenase mean value. It was concluded that Increased activity of all the serum enzymes was due to modulation of liver functions directly or indirectly.

  14. Heat stress induced changes in metabolic regulators of donkeys from arid tracts in India

    Directory of Open Access Journals (Sweden)

    Kataria N.

    2012-05-01

    Full Text Available To find out heat stress induced changes in metabolic regulators of donkeys from arid tracts in India, blood samples were collected to harvest the serum during moderate and extreme hot ambiences. The metabolic enzymes determined were sorbitol dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase, gammaglutamayl transferase, 5’nucleotidase, glucose-6-phosphatase, arginase, and aldolase. The mean values of all the serum enzymes increased significantly (p≤0.05 during hot ambience as compared to respective values during moderate ambience. It was concluded that increased activity of all the enzymes in the serum was due to modulation of metabolic reactions to combat the effect of hot ambience on the animals. Activation of gluconeogenesis along with hexose monophosphate shunt and urea cycle probably helped the animals to combat the heat stress.

  15. The Effect of 5'-Adenylic Acid on Hepatic Proteome of Mice Radiated by 60Co γ-ray

    Directory of Open Access Journals (Sweden)

    Cuilin Cheng

    2013-12-01

    Full Text Available Understanding the protection mechanism of 5'-AMP requires comprehensive knowledge of the proteins expressed during the period that the body is exposed to irradiation. Proteomics provides the tools for such analyses. Here, the experimental ICR mice were divided into three groups (normal group, model group and 5'-AMP + irradiation group. After different treatment, the hepatic total protein of each animal in three groups was separated by two-dimensional gel electrophoresis (2-DE. 2-DE analysis revealed fifty-eight protein spots were differentially expressed in comparison to three groups. From 58 protein spots, we selected nine spots to identify by MALDI-TOF-MS and received credible results. They were determined to be type I arginase, annexin A5, regucalcin, catalase, Tpm3 protein, Pdia4 protein, 14-3-3 protein epsilon, NAD-Malate dehydrogenase and heat shock protein 90. Considering the characteristic of these proteins, we proposed a possible protection pathway.

  16. RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes.

    Science.gov (United States)

    Foldi, Julia; Shang, Yingli; Zhao, Baohong; Ivashkiv, Lionel B; Hu, Xiaoyu

    2016-03-01

    Development of alternatively activated (M2) macrophage phenotypes is a complex process that is coordinately regulated by a plethora of pathways and factors. Here, we report that RBP-J, a DNA-binding protein that integrates signals from multiple pathways including the Notch pathway, is critically involved in polarization of M2 macrophages. Mice deficient in RBP-J in the myeloid compartment exhibited impaired M2 phenotypes in vivo in a chitin-induced model of M2 polarization. Consistent with the in vivo findings, M2 polarization was partially compromised in vitro in Rbpj-deficient macrophages as demonstrated by reduced expression of a subset of M2 effector molecules including arginase 1. Functionally, myeloid Rbpj deficiency impaired M2 effector functions including recruitment of eosinophils and suppression of T cell proliferation. Collectively, we have identified RBP-J as an essential regulator of differentiation and function of alternatively activated macrophages.

  17. IMMUNOSUPPRESSIVE EFFECTS OF ARGININE DEIMINASE FROM STREPTOCOCCUS PYOGENES

    Directory of Open Access Journals (Sweden)

    E. A. Starikova

    2015-01-01

    Full Text Available Many pathogens use metabolic pathway of arginine for successful dissemination. Bacterial arginine deiminase hydrolyzes arginine to form one molecule of ammonia and two molecules of ATP. The activity of the enzyme contributes to the improvement of survival of pathogenic bacteria in conditions of low pH at the site of infection or in phagolysosome, as well as in anaerobic conditions, and also leads to deficiency of arginine. Metabolism of arginine plays an important role in regulating the functions of immune system cells in mammals. Arginine is a substrate of enzymes NOS and arginase. Arginine depletion, potentially contributs to immunosuppression. The review analyzed the literature data on the effect of streptococcal arginine deiminase on the metabolism of arginine eukaryotic cells, and discusses immunosuppressive action of the enzyme.

  18. [Effect of Arnica montana tincture on some hydrolytic enzyme activities of rat liver in experimental toxic hepatitis].

    Science.gov (United States)

    Iaremiĭ, I M; Meshchyshen, I F; Hrihor'ieva, N P; Kostiuk, L S

    1998-01-01

    Effects of tinctura arnica on arginase, adenosine triphosphatase, glucose-6-phosphatase and 5'-nucleotidase activities of rats liver in case of experimental toxic hepatitis have been studied. Toxic hepatitis was caused by 2 times interstomach administration of 0.25 ml oil solution of carbon tetrachloride per 100 g of animal weight. 20 mkl/100 g of tinctura arnica was administered every day per os for 14 days. The enzyme activities have been investigated at 3, 7 and 17 days. A significant demention of a studied hydrolytic enzyme activities in rats liver at intoxication of the body by CCI4 has been shown. It has been established that tinctura arnica administered per os to intoxicated animals sped up the normalization of hydrolytic enzyme activities in rat liver.

  19. Synergistic effects between catalase inhibitors and modulators of nitric oxide metabolism on tumor cell apoptosis.

    Science.gov (United States)

    Scheit, Katrin; Bauer, Georg

    2014-10-01

    Inhibitors of catalase (such as ascorbate, methyldopa, salicylic acid and neutralizing antibodies) synergize with modulators of nitric oxide (NO) metabolism (such as arginine, arginase inhibitor, NO synthase-inducing interferons and NO dioxygenase inhibitors) in the singlet oxygen-mediated inactivation of tumor cell protective catalase. This is followed by reactive oxygen species (ROS)-dependent apoptosis induction. TGF-beta, NADPH oxidase-1, NO synthase, dual oxidase-1 and caspase-9 are characterized as essential catalysts in this process. The FAS receptor and caspase-8 are required for amplification of ROS signaling triggered by individual compounds, but are dispensable when the synergistic effect is established. Our findings explain the antitumor effects of catalase inhibitors and of compounds that target NO metabolism, as well as their synergy. These data may have an impact on epidemiological studies related to secondary plant compounds and open new perspectives for the establishment of novel antitumor drugs and for the improvement of established chemotherapeutics.

  20. Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

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    Hiromu Suzuki

    2014-07-01

    Full Text Available The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1 infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8.

  1. Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis

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    Jaffrezic-Renault N.

    2012-12-01

    Full Text Available Aim. To determine an influence of serum components on the L-arginine biosensor sensitivity and to formulate practical recommendations for its reliable analysis. Methods. The L-arginine biosensor comprised arginase and urease co-immobilized by cross-linking. Results. The biosensor specificity was investigated based on a series of representative studies (namely, through urea determination in the serum; inhibitory effect studies of mercury ions; high temperature treatment of sensors; studying the biosensor sensitivity to the serum treated by enzymes, and selectivity studies. It was found that the response of the biosensor to the serum injections was determined by high sensitivity of the L-arginine biosensor toward not only to L-arginine but also toward two other basic amino acids (L-lysine and L-histidine. Conclusions. A detailed procedure of optimization of the conductometric biosensor for L-arginine determination in blood serum has been proposed.

  2. Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts.

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    Fontaniella, Blanca; Millanes, Ana-María; Vicente, Carlos; Legaz, María-Estrella

    2004-12-01

    Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn(2+). The cation cannot be substituted by Ca(2+) which, in addition, inhibits Mn(2+)-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease.

  3. Changes of nitric oxide system and lipid peroxidation parameters in the digestive system of rats under conditions of acute stress, and use of nonsteroidal anti-inflammatory drugs

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    Fomenko Iryna

    2015-03-01

    Full Text Available The use of nonsteroidal anti-inflammatory drugs (NSAIDs in combination with being physiologically stressed often occurs in in the course of different pathologies. This situation may result in the alteration of digestive system functioning. The effect of stress brings about changes in the activity of nitric oxide synthase (NOS, arginase, cyclooxygenase (COX and lipid peroxidation, whereas the use of NSAIDs interrupts the multiple functions of the cell via the inhibition of prostaglandins (PGs synthesis. Taking into account that NOS and COX-systems are connected in their regulation, the aim of the study was to determine the role played by NOS and lipid peroxidation under conditions of the combined action of NSAIDs and stress. In our study, male rats were used. The NSAIDs (naproxen - a non-selective COX inhibitor, celecoxib - a selective COX-2 blocker, and the compound 2A5DHT (which is the active substance of dual COX, and the lipoxygenase (LOX inhibitor, darbufelone were all administered at a dose 10 mg/kg, prior to water restraint stress (WRS. WRS brought about an increase of inducible NOS (iNOS activity in the intestinal mucosal and muscular membranes, as well as in the pancreas. Because of this, constitutive NOS izoform (cNOS and arginase activities decreased. Moreover, the MDA concentration increased, indicating the development of oxidative stress. In our work, pretreatment with naproxen, as in the WRS model, engendered a decrease in iNOS activity. What is more, administration of Celecoxib did not change iNOS activity, as compared to WRS alone, and it showed a tendency to reduce lipid peroxidation. In addition, 2A5DHT prior WRS brought about a decrease of iNOS activity, with the subsequent rise of cNOS activity. Of note, MDA concentration decreased in all studied organs, indicating the reduction of lipid peroxidation under the action of the darbufelone active substance.

  4. Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

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    Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

    2008-12-01

    The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

  5. An orthotopic xenograft model with survival hindlimb amputation allows investigation of the effect of tumor microenvironment on sarcoma metastasis.

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    Goldstein, Seth D; Hayashi, Masanori; Albert, Catherine M; Jackson, Kyle W; Loeb, David M

    2015-10-01

    Overall survival rates for pediatric high-grade sarcoma have improved greatly in the past few decades, but prevention and treatment of distant metastasis remain the most compelling problems facing these patients. Traditional preclinical mouse models have not proven adequate to study the biology and treatment of spontaneous distant sarcoma metastasis. To address this deficit, we developed an orthotopic implantation/amputation model in which patient-derived sarcoma xenografts are surgically implanted into mouse hindlimbs, allowed to grow, then subsequently amputated and the animals observed for development of metastases. NOD/SCID/IL-2Rγ-null mice were implanted with either histologically intact high grade sarcoma patient-derived xenografts or cell lines in the pretibial space and affected limbs were amputated after tumor growth. In contrast to subcutaneous flank tumors, we were able to consistently detect spontaneous distant spread of the tumors using our model. Metastases were seen in 27-90 % of animals, depending on the xenograft, and were repeatable and predictable. We also demonstrate the utility of this model for studying the biology of metastasis and present preliminary new insights suggesting the role of arginine metabolism and macrophage phenotype polarization in creating a tumor microenvironment that facilitates metastasis. Subcutaneous tumors express more arginase than inducible nitric oxide synthase and demonstrate significant macrophage infiltration, whereas orthotopic tumors express similar amounts of inducible nitric oxide synthase and arginase and have only a scant macrophage infiltrate. Thus, we present a model of spontaneous distant sarcoma metastasis that mimics the clinical situation and is amenable to studying the biology of the entire metastatic cascade.

  6. Macrophage plasticity in experimental atherosclerosis.

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    Jamila Khallou-Laschet

    Full Text Available As in human disease, macrophages (MØ are central players in the development and progression of experimental atherosclerosis. In this study we have evaluated the phenotype of MØ associated with progression of atherosclerosis in the apolipoprotein E (ApoE knockout (KO mouse model.We found that bone marrow-derived MØ submitted to M1 and M2 polarization specifically expressed arginase (Arg II and Arg I, respectively. This distinct arginase expression was used to evaluate the frequency and distribution of M1 and M2 MØ in cross-sections of atherosclerotic plaques of ApoE KO mice. Early lesions were infiltrated by Arg I(+ (M2 MØ. This type of MØ favored the proliferation of smooth muscle cells, in vitro. Arg II(+ (M1 MØ appeared and prevailed in lesions of aged ApoE KO mice and lesion progression was correlated with the dominance of M1 over the M2 MØ phenotype. In order to address whether the M2->M1 switch could be due to a phenotypic switch of the infiltrated cells, we performed in vitro repolarization experiments. We found that fully polarized MØ retained their plasticity since they could revert their phenotype. The analysis of the distribution of Arg I- and Arg II-expressing MØ also argued against a recent recruitment of M1 MØ in the lesion. The combined data therefore suggest that the M2->M1 switch observed in vivo is due to a conversion of cells already present in the lesion. Our study suggests that interventional tools able to revert the MØ infiltrate towards the M2 phenotype may exert an atheroprotective action.

  7. Plaque Size Is Decreased but M1 Macrophage Polarization and Rupture Related Metalloproteinase Expression Are Maintained after Deleting T-Bet in ApoE Null Mice.

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    Aikaterini Tsaousi

    Full Text Available Thelper1 (Th1 lymphocytes have been previously implicated in atherosclerotic plaque growth but their role in plaque vulnerability to rupture is less clear. We investigated whether T-bet knockout that prevents Th1 lymphocyte differentiation modulates classical (M1 macrophage activation or production of matrix degrading metalloproteinases (MMPs and their tissue inhibitors, TIMPs.We studied the effect of T-bet deletion in apolipoproteinE (ApoE knockout mice fed a high fat diet (HFD or normal chow diet (ND. Transcript levels of M1/M2 macrophage polarization markers, selected MMPs and TIMPs were measured by RT-qPCR in macrophages isolated from subcutaneous granulomas or in whole aortae. Immunohistochemistry of aortic sinus (AS and brachiocephalic artery (BCA plaques was conducted to quantify protein expression of the same factors. Deletion of T-bet decreased mRNA for the M1 marker NOS-2 in granuloma macrophages but levels of M2 markers (CD206, arginase-1 and Ym-1, MMPs-2, -9, -12, -13, -14 and -19 or TIMPs-1 to -3 were unchanged. No mRNA differences were observed in aortic extracts from mice fed a HFD for 12 weeks. Moreover, AS and BCA plaques were similarly sized between genotypes, and had similar areas stained for NOS-2, COX-2, MMP-12 and MMP-14 proteins. T-bet deletion increased MMP-13, MMP-14 and arginase-1 in AS plaques. After 35 weeks of ND, T-bet deletion reduced the size of AS and BCA plaques but there were no differences in the percentage areas stained for M1 or M2 markers, MMPs-12, -13, -14, or TIMP-3.Absence of Th1 lymphocytes is associated with reduced plaque size in ApoE knockout mice fed a normal but not high fat diet. In either case, M1 macrophage polarization and expression of several MMPs related to plaque instability are either maintained or increased.

  8. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages.

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    Crystal Rocher

    Full Text Available Previously we demonstrated that bone morphogenetic protein-7 (BMP-7 treatment polarizes monocytes into M2 macrophages and increases the expression of anti-inflammatory cytokines. Despite these findings, the mechanisms for the observed BMP-7 induced monocyte polarization into M2 macrophages are completely unknown. In this study, we demonstrate the mechanisms involved in the polarization of monocytes into M2 macrophages. Apoptotic conditioned media (ACM was generated to mimic the stressed conditions, inducing monocyte polarization. Monocytes were treated with ACM along with BMP-7 and/or its inhibitor, follistatin, for 48 hours. Furthermore, an inhibitor of the PI3K pathway, LY-294002, was also studied. Our data show that BMP-7 induces polarization of monocytes into M2 macrophages while significantly increasing the expression of anti-inflammatory markers, arginase-1 and IL-10, and significantly (p<0.05 decreasing the expression of pro-inflammatory markers iNOS, IL-6, TNF-α and MCP-1; (p<0.05. Moreover, addition of the PI3K inhibitor, LY-294002, significantly (p<0.05 decreases upregulation of IL-10 and arginase-1, suggesting involvement of the PI3K pathway in M2 macrophage polarization. Next, following BMP-7 treatment, a significant (p<0.05 increase in p-SMAD1/5/8 and p-PI3K expression resulting in downstream activation of p-Akt and p-mTOR was observed. Furthermore, expression of p-PTEN, an inhibitor of the PI3K pathway, was significantly (p<0.05 increased in the ACM group. However, BMP-7 treatment inhibited its expression, suggesting involvement of the PI3K-Akt-mTOR pathway. In conclusion, we demonstrate that BMP-7 polarizes monocytes into M2 macrophages and enhances anti-inflammatory cytokine expression which is mediated by the activated SMAD-PI3K-Akt-mTOR pathway.

  9. Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

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    David J. Tate Jr., John R. Patterson, Cruz Velasco-Gonzalez, Emily N. Carroll, Janie Trinh, Daniel Edwards, Ashok Aiyar, Beatriz Finkel-Jimenez, Arnold H. Zea

    2012-01-01

    Full Text Available Renal cell carcinoma (RCC remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ, indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS protein, resulting in a sustained elevation of nitric oxide (NO and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.

  10. Human Mesenchymal Stem Cell-Educated Macrophages Are a Distinct High IL-6-Producing Subset that Confer Protection in Graft-versus-Host-Disease and Radiation Injury Models.

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    Bouchlaka, Myriam N; Moffitt, Andrea B; Kim, Jaehyup; Kink, John A; Bloom, Debra D; Love, Cassandra; Dave, Sandeep; Hematti, Peiman; Capitini, Christian M

    2017-02-28

    Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to prevent or treat graft-versus-host disease (GVHD) have shown mixed results. Macrophages (MØs) are important regulators of immunity and can promote tissue regeneration and remodeling. We have previously shown that MSCs can educate MØs toward a unique anti-inflammatory immunophenotype (MSC-educated macrophages [MEMs]); however, their implications for in vivo models of inflammation have not been studied yet. We now show that in comparison with MØs, MEMs have increased expression of the inhibitory molecules PD-L1, PD-L2, in addition to markers of alternatively activated macrophages: CD206 and CD163. RNA-Seq analysis of MEMs, as compared with MØs, show a distinct gene expression profile that positively correlates with multiple pathways important in tissue repair. MEMs also show increased expression of IL-6, transforming growth factor-β, arginase-1, CD73, and decreased expression of IL-12 and tumor necrosis factor-α. We show that IL-6 secretion is controlled in part by the cyclo-oxygenase-2, arginase, and JAK1/STAT1 pathway. When tested in vivo, we show that human MEMs significantly enhance survival from lethal GVHD and improve survival of mice from radiation injury. We show these effects could be mediated in part through suppression of human T cell proliferation and may have attenuated host tissue injury in part by enhancing murine fibroblast proliferation. MEMs are a unique MØ subset with therapeutic potential for the management of GVHD and/or protection from radiation-induced injury.

  11. Macrophage Infiltration and Alternative Activation during Wound Healing Promote MEK1-Induced Skin Carcinogenesis.

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    Weber, Christine; Telerman, Stephanie B; Reimer, Andreas S; Sequeira, Ines; Liakath-Ali, Kifayathullah; Arwert, Esther N; Watt, Fiona M

    2016-02-15

    Macrophages are essential for the progression and maintenance of many cancers, but their role during the earliest stages of tumor formation is unclear. To test this, we used a previously described transgenic mouse model of wound-induced skin tumorigenesis, in which expression of constitutively active MEK1 in differentiating epidermal cells results in chronic inflammation (InvEE mice). Upon wounding, the number of epidermal and dermal monocytes and macrophages increased in wild-type and InvEE skin, but the increase was greater, more rapid, and more sustained in InvEE skin. Macrophage ablation reduced tumor incidence. Furthermore, bioluminescent imaging in live mice to monitor macrophage flux at wound sites revealed that macrophage accumulation was predictive of tumor formation; wounds with the greatest number of macrophages at day 5 went on to develop tumors. Gene expression profiling of flow-sorted monocytes, macrophages, and T cells from InvEE and wild-type skin showed that as wound healing progressed, InvEE macrophages altered their phenotype. Throughout wound healing and after wound closure, InvEE macrophages demonstrated sustained upregulation of several markers implicated in alternative macrophage activation including arginase-1 (ARG1) and mannose receptor (CD206). Notably, inhibition of ARG1 activity significantly reduced tumor formation and epidermal proliferation in vivo, whereas addition of L-arginase to cultured keratinocytes stimulated proliferation. We conclude that macrophages play a key role in early, inflammation-mediated skin tumorigenesis, with mechanistic evidence suggesting that ARG1 secretion drives tumor development by stimulating epidermal cell proliferation. These findings highlight the importance of cancer immunotherapies aiming to polarize tumor-associated macrophages toward an antitumor phenotype.

  12. Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in channel catfish macrophages.

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    Baumgartner, Wes A; Dubytska, Lidiya; Rogge, Matthew L; Mottram, Peter J; Thune, Ronald L

    2014-06-01

    Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H(+)-ATPases by use of the specific inhibitor bafilomycin A1 abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.

  13. The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

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    Ruijter Jan M

    2008-11-01

    Full Text Available Abstract Background Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS, ornithine aminotransferase (OAT, argininosuccinate synthetase (ASS, arginase-1 (ARG1, arginase-2 (ARG2, and nitric-oxide synthase (NOS were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Results Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. Conclusion The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

  14. Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

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    Kashyap, Sonu; Warner, Gina M; Hartono, Stella P; Boyilla, Rajendra; Knudsen, Bruce E; Zubair, Adeel S; Lien, Karen; Nath, Karl A; Textor, Stephen C; Lerman, Lilach O; Grande, Joseph P

    2016-03-01

    Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

  15. Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection

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    Elahi, Shokrollah; Ertelt, James M.; Kinder, Jeremy M.; Jiang, Tony T.; Zhang, Xuzhe; Xin, Lijun; Chaturvedi, Vandana; Strong, Beverly S.; Qualls, Joseph E.; Steinbrecher, Kris A.; Kalfa, Theodosia A.; Shaaban, Aimen F.; Way, Sing Sing

    2013-12-01

    Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71+ erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71+ cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71+ cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71+ cell-mediated susceptibility to infection is counterbalanced by CD71+ cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71+ cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these

  16. Systemic and Cardiac Depletion of M2 Macrophage through CSF-1R Signaling Inhibition Alters Cardiac Function Post Myocardial Infarction.

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    Leblond, Anne-Laure; Klinkert, Kerstin; Martin, Kenneth; Turner, Elizebeth C; Kumar, Arun H; Browne, Tara; Caplice, Noel M

    2015-01-01

    The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1lo and F4/80hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1β), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.

  17. Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

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    Burnat, Mireia; Flores, Enrique

    2014-10-01

    Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

  18. Persistent Coxiella burnetii infection in mice overexpressing IL-10: an efficient model for chronic Q fever pathogenesis.

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    Soraya Meghari

    2008-02-01

    Full Text Available Interleukin (IL-10 increases host susceptibility to microorganisms and is involved in intracellular persistence of bacterial pathogens. IL-10 is associated with chronic Q fever, an infectious disease due to the intracellular bacterium Coxiella burnetii. Nevertheless, accurate animal models of chronic C. burnetii infection are lacking. Transgenic mice constitutively expressing IL-10 in macrophages were infected with C. burnetti by intraperitoneal and intratracheal routes and infection was analyzed through real-time PCR and antibody production. Transgenic mice exhibited sustained tissue infection and strong antibody response in contrast to wild-type mice; thus, bacterial persistence was IL-10-dependent as in chronic Q fever. The number of granulomas was low in spleen and liver of transgenic mice infected through the intraperitoneal route, as in patients with chronic Q fever. Macrophages from transgenic mice were unable to kill C. burnetii. C. burnetii-stimulated macrophages were characterized by non-microbicidal transcriptional program consisting of increased expression of arginase-1, mannose receptor, and Ym1/2, in contrast to wild-type macrophages in which expression of inducible NO synthase and inflammatory cytokines was increased. In vivo results emphasized macrophage data. In spleen and liver of transgenic mice infected with C. burnetii by the intraperitoneal route, the expression of arginase-1 was increased while microbicidal pathway consisting of IL-12p40, IL-23p19, and inducible NO synthase was depressed. The overexpression of IL-10 in macrophages prevents anti-infectious competence of host, including the ability to mount granulomatous response and microbicidal pathway in tissues. To our knowledge, this is the first efficient model for chronic Q fever pathogenesis.

  19. First evidence of intraclonal genetic exchange in trypanosomatids using two Leishmania infantum fluorescent transgenic clones.

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    Estefanía Calvo-Álvarez

    2014-09-01

    Full Text Available The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum.We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains.A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.

  20. Pomegranate juice polyphenols induce a phenotypic switch in macrophage polarization favoring a M2 anti-inflammatory state.

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    Aharoni, Saar; Lati, Yoni; Aviram, Michael; Fuhrman, Bianca

    2015-01-01

    It was documented that pomegranate has anti-inflammatory effects. In this study, we investigated a direct effect of pomegranate juice (PJ) and its polyphenols on macrophage inflammatory phenotype. In vitro, PJ and its major polyphenols dose-dependently attenuated macrophage response to M1 proinflammatory activation in J774.A1 macrophage-like cell line. This was evidenced by a significant decrease in TNFα and IL-6 secretion in response to stimulation by IFNγ and Lipopolysaccharide. In addition, PJ and punicalagin dose-dependently promoted the macrophages toward a M2 anti-inflammatory phenotype, as determined by a significant increase in the spontaneous secretion of IL-10. In mice, supplementation with dietary PJ substantially inhibited the M2 to M1 macrophage phenotypic shift associated with age, toward a favorable anti-inflammatory M2 phenotype. This effect was also reflected in the mice atherosclerotic plaques, as evaluated by the distinct expression of arginase isoforms. PJ consumption inhibited the increment of arginase II (Arg II, M1) mRNA expression during aging, and maintained the levels of Arg I (M2) expression similar to those in young mice aorta. This study demonstrates, for the first time, that pomegranate polyphenols directly suppress macrophage inflammatory responses and promote M1 to M2 switch in macrophage phenotype. Furthermore, this study indicates that PJ consumption may inhibit the progressive proinflammatory state in the aorta along atherosclerosis development with aging, due to a switch in macrophage phenotype from proinflammatory M1 to anti-inflammatory M2.

  1. Dietary supplementation of ginger and turmeric improves reproductive function in hypertensive male rats

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    Ayodele Jacob Akinyemi

    2015-01-01

    Full Text Available Ginger [Zingiber officinale Roscoe (Zingiberaceae] and turmeric [Curcuma longa Linn (Zingiberaceae] rhizomes have been reportedly used in folk medicine for the treatment of hypertension. However, the prevention of its complication such as male infertility remains unexplored. Hence, the aim of the present study was to investigate the preventive effects of ginger and turmeric rhizomes on some biomarkers of male reproductive function in L-NAME-induced hypertensive rats. Male Wistar rats were divided into seven groups (n = 10: normotensive control rats; induced (L-NAME hypertensive rats; hypertensive rats treated with atenolol (10 mg/kg/day; normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of L-NAME (40 mg/kg/day. The results revealed significant decrease in serum total testosterone and epididymal sperm progressive motility without affecting sperm viability in hypertensive rats. Moreover, increased oxidative stress in the testes and epididymides of hypertensive rats was evidenced by significant decrease in total and non-protein thiol levels, glutathione S-transferase (GST activity with concomitant increase in 2′,7′-dichlorofluorescein (DFCH oxidation and thiobarbituric acid reactive substances (TBARS production. Similarly, decreased testicular and epididymal NO level with concomitant elevation in arginase activity was observed in hypertensive rats. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations in biomarkers of reproductive function in hypertensive rats. The inhibition of arginase activity and increase in NO and testosterone levels by both rhizomes could suggest possible mechanism of action for the prevention of male infertility in hypertension. Therefore, both rhizomes could be harnessed as functional foods to prevent hypertension

  2. IFN-γ differentially regulates subsets of Gr-1(+)CD11b(+) myeloid cells in chronic inflammation.

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    Zhan, Xiaoxia; Fang, Yimin; Hu, Shengfeng; Wu, Yongjian; Yang, Kun; Liao, Chunxin; Zhang, Yuanqing; Huang, Xi; Wu, Minhao

    2015-08-01

    During chronic inflammation, prolonged over-reactive immune response may lead to tissue destruction, while immune suppression hinders tissue repair and pathogen elimination. Therefore, precise regulation of the immune response is needed to avoid immuno-pathology. Interferon-gamma (IFN-γ) is widely used in clinical treatment of inflammatory diseases. However, the underlying mechanism remains unclear. Here, we evaluated the role of IFN-γ on CD11b(+)Gr-1(+) myeloid cell differentiation and function, using a heat-killed Mycobacterium bovis BCG-induced chronic inflammation model. After challenge with heat-killed BCG, two subpopulations of CD11b(+)Gr-1(+) myeloid cells were generated in the mouse spleen. Phenotypical, morphological and functional analysis indicated that the CD11b(+)Gr-1(high) Ly6G(high) Ly6C(low) subset was neutrophil-like myeloid-derived inducer cells (N-MDICs), which promoted T cell activation, while the other subset was CD11b(+)Gr-1(low) Ly6G(neg) Ly6C(high) monocyte-like myeloid-derived suppressor cells (M-MDSCs) that displayed extensive suppressor function. IFN-γ treatment dampened N-MDICs-mediated T cell activation through up-regulating T cell suppressive mediators, reactive oxygen species (ROS) and arginase I. While for M-MDSCs, IFN-γ reduced their suppressing activity by decreasing the arginase activity. Our study provides evidence that IFN-γ balances the over-reactive vs compromised immune response through different regulation of distinct myeloid subsets, and therefore displays significant therapeutic potential for effective immuno-therapy of chronic inflammatory diseases.

  3. IL-4对肝片吸虫感染诱导的巨噬细胞中脂肪酸结合蛋白2分布的影响%Effects of IL-4 on Distribution of FABP2 in Fasciola hepatica Elicited Macrophages

    Institute of Scientific and Technical Information of China (English)

    王晶; 张文韬; 王豪举; 郭智莉; 周雪梅; 周容琼; 周作勇; 罗洪林

    2012-01-01

    为阐明小鼠感染肝片吸虫后巨噬细胞中脂肪酸结合蛋白2 (FABP2)的分布,采用肝片吸虫囊蚴为感染源,经口分别感染雌性BALB/c野生型(WT)及其IL-4单抗处置小鼠,在运用特异性PCR鉴定成功感染小鼠后获取巨噬细胞,并设立人工巯基醋酸盐诱导巨噬细胞对照组进行体外培养.用荧光定量PCR检测选择性激活巨噬细胞的标记蛋白Relmα、Ym1和Arginase1以确定其激活状态.采用特异性抗体对所获取的巨噬细胞细胞质染色后用共聚焦显微镜摄取不同时间点从不同小鼠体内获取的巨噬细胞染色后的图片.野生型BALB/c巨噬细胞中Relma、Ym1和Arginase1均大量表达;IL-4单抗处置BALB/c小鼠和巯基醋酸盐诱导小鼠中巨噬细胞中的Relmα、Ym1和Arginase1的表达量较野生型小鼠中的极显著或显著下降.FABP2在FeMΦ中并不分布于细胞质膜下,而是呈点状广泛分布于细胞质中,其荧光强度在12~24 h间呈上升趋势,在24~48 h间却呈下降趋势.%Macrophages were acquired from the F. hepatica metacercariae infected IL-4 monoclonal antibody treated and wild type female BALB/c mice, respectively. All F. hepatica elicited macrophages and control macrophages were cultured in vitro. mRNAs from macrophages were extracted for the detection of Relma, Ym1 and Arginasel genes using Real-time poly-merase chain reaction. Pictures and fluorescence intensity at different time points were acquired with a confocal microscopy and image software respectively after staining with serial purified antibodies. Results showed that levels of Relmα, Yml and Arginasel in F. hepatica elicited macrophages (FeMΦ) and control macrophages (TeMΦ) from IL-4 monoclonal antibody treated mice were significantly decreased comparison to that in wild type mice. FABP2 was detected as spots in the cytoplasm. The fluorescence intensity of FABP2 increased from 12 to 24 h, but decreased afterward to 48 h. The fluorescence intensity of

  4. SOCS3基因敲除促进小鼠脊髓损伤处巨噬细胞向M2型分化%SOCS3 conditional ablation induces differentiation of M2-type macrophages after spinal cord injury in mice

    Institute of Scientific and Technical Information of China (English)

    党圆圆; 张洪钿; 籍新潮; 吴翠莹; 陈晨; 杨艺; 徐如祥

    2016-01-01

    目的 探讨条件性敲除细胞因子信号转导抑制蛋白3(SOCS3)基因后小鼠脊髓损伤处巨噬细胞表型变化及运动功能恢复情况. 方法 以神经系统条件性敲除SOCS3基因的基因工程小鼠(Socs3fVfl Nes cre)(实验组)及未敲除SOCS3基因的同窝小鼠(Socs3fl)(对照组)为研究对象,每组40只.2组小鼠分别各取35只对T10节段脊髓施以钳夹伤,另5只仅做假手术对照.每组分别取15只损伤模型小鼠在术后3、7、14d用于免疫荧光染色检测Arginase1(巨噬细胞M2a型、M2c型表型标志物)及CD86(巨噬细胞M1型、M2b型表型标志物)表达;每组分别取剩余20只损伤模型小鼠在术后1、3、7、14d用于荧光定量RT-PCR检测Arginase1、CD206(巨噬细胞M2型表型标志物)、诱导型一氧化氮合酶(iNOS)、CD32(巨噬细胞M1型表型标志物)及SOCS3、信号传导与转录激活因子3(STAT3)基因表达,并以假手术对照小鼠为基线;在术后1、3、7、14d采用BMS量表评价小鼠后肢及躯干运动功能. 结果 行为学实验结果显示:实验组小鼠BMS评分在术后3、7、14d明显高于对照组,差异均有统计学意义(P<0.05).对Iba1与CD86及Arginase1共染色细胞进行计数半定量分析发现:实验组中Iba1与Arginase1共染色阳性细胞在术后3、7、14d明显多于对照组,差异均有统计学意义(P<0.05);实验组中Iba1与CD86共染色阳性细胞在术后7、14d明显少于对照组,差异均有统计学意义(P<0.05).荧光定量PCR结果显示:术后1、3d,实验组SOCS3基因表达量较对照组明显减低,差异均有统计学意义(P<0.05).术后3、7、14d,实验组STA T3基因表达量较对照组明显增高,差异均有统计学意义(P<0.05).与对照组相比,术后7d实验组Arginase1及CD206基因表达量明显增加,差异均有统计学意义(P<0.05);与对照组相比,术后14d实验组CD206基因表达量明显增加,CD32基因表达量明显降低,差异均有统计学意义(P<0

  5. Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis

    Science.gov (United States)

    Carneiro, Priscila J.; Clevelario, Amanda L.; Padilha, Gisele A.; Silva, Johnatas D.; Kitoko, Jamil Z.; Olsen, Priscilla C.; Capelozzi, Vera L.; Rocco, Patricia R. M.; Cruz, Fernanda F.

    2017-01-01

    Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1β, TNF-α, IFN-γ, and TGF-β levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph

  6. Modulation of functional characteristics of resident and thioglycollate-elicited peritoneal murine macrophages by a recombinant banana lectin

    Science.gov (United States)

    Marinkovic, Emilija; Djokic, Radmila; Lukic, Ivana; Filipovic, Ana; Inic-Kanada, Aleksandra; Kosanovic, Dejana; Gavrovic-Jankulovic, Marija; Stojanovic, Marijana

    2017-01-01

    We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and physiological impacts highly resemble those reported for its natural counterparts, binds murine peritoneal macrophages and specifically modulates their functional characteristics. By using rBanLec in concentrations ranging from 1 μg to 10 μg to stimulate resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages from BALB/c and C57BL/6 mice, we have shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages and their genetic background. rBanLec, in a positive dose-dependent manner, promotes the proliferation of TGMs from both BALB/c and C57BL/6 mice, while its mitogenic influence on RMs is significantly lower (BALB/c mice) or not detectable (C57BL/6 mice). In all peritoneal macrophages, irrespective of their type and genetic background, rBanLec, in a positive dose dependent manner, enhances the secretion of IL-10. rBanLec stimulation of RMs from both BALB/c and C57BL/6 resulted in a positive dose-dependent promotion of proinflammatory phenotype (enhancement of NO production and IL-12 and TNFα secretion, reduction of arginase activity). Positive dose-dependent skewing toward proinflammatory phenotype was also observed in TGMs from C57BL/6 mice. However, the enhancement of rBanLec stimulation promotes skewing of TGMs from BALB/c mice towards anti-inflammatory profile (reduction of NO production and IL-12 secretion, enhancement of arginase activity and TGFβ and IL-4 secretion). Moreover, we established that rBanLec binds oligosaccharide structures of TLR2 and CD14 and that blocking of signaling via these receptors significantly impairs the production of TNFα and NO in BALB/c macrophages. Since the outcome of rBanLec stimulation depends on rBanLec concentration as well as on the functional characteristics of its target cells and their genetic background, further studies are needed to investigate

  7. Role of myeloid-derived suppressor cells in amelioration of experimental autoimmune hepatitis following activation of TRPV1 receptors by cannabidiol.

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    Venkatesh L Hegde

    Full Text Available BACKGROUND: Myeloid-derived suppressor cells (MDSCs are getting increased attention as one of the main regulatory cells of the immune system. They are induced at sites of inflammation and can potently suppress T cell functions. In the current study, we demonstrate how activation of TRPV1 vanilloid receptors can trigger MDSCs, which in turn, can inhibit inflammation and hepatitis. METHODOLOGY/PRINCIPAL FINDINGS: Polyclonal activation of T cells, following injection of concanavalin A (ConA, in C57BL/6 mice caused acute hepatitis, characterized by significant increase in aspartate transaminase (AST, induction of inflammatory cytokines, and infiltration of mononuclear cells in the liver, leading to severe liver injury. Administration of cannabidiol (CBD, a natural non-psychoactive cannabinoid, after ConA challenge, inhibited hepatitis in a dose-dependent manner, along with all of the associated inflammation markers. Phenotypic analysis of liver infiltrating cells showed that CBD-mediated suppression of hepatitis was associated with increased induction of arginase-expressing CD11b(+Gr-1(+ MDSCs. Purified CBD-induced MDSCs could effectively suppress T cell proliferation in vitro in arginase-dependent manner. Furthermore, adoptive transfer of purified MDSCs into naïve mice conferred significant protection from ConA-induced hepatitis. CBD failed to induce MDSCs and suppress hepatitis in the livers of vanilloid receptor-deficient mice (TRPV1(-/- thereby suggesting that CBD primarily acted via this receptor to induce MDSCs and suppress hepatitis. While MDSCs induced by CBD in liver consisted of granulocytic and monocytic subsets at a ratio of ∼2∶1, the monocytic MDSCs were more immunosuppressive compared to granulocytic MDSCs. The ability of CBD to induce MDSCs and suppress hepatitis was also demonstrable in Staphylococcal enterotoxin B-induced liver injury. CONCLUSIONS/SIGNIFICANCE: This study demonstrates for the first time that MDSCs play a

  8. L-citrulline protects from kidney damage in type 1 diabetic mice.

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    Maritza J Romero

    2013-12-01

    Full Text Available Rationale. Diabetic nephropathy is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of L-arginine (L-arg, the substrate for endothelial nitric oxide synthase (eNOS, failed to improve vascular function. L-citrulline (L-cit supplementation not only increases L-arg synthesis, but also inhibits cytosolic arginase I (Arg I, a competitor of eNOS for the use of L-arg, in the vasculature. Aims. To investigate whether L-cit treatment reduces diabetic nephropathy in streptozotocin (STZ-induced type 1 diabetes in mice and rats and to study its effects on arginase II (ArgII function, the main renal isoform. Methods. STZ-C57BL6 mice received L-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and L-cit-treated STZ-rats were evaluated. Results. L-cit exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, L-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 wks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater BUN levels, hypertrophy, and dilated tubules than diabetic wild type mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic wild type animals. L-cit also restored NO/ROS balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, L-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1beta and IL-12(p70 generation in the human proximal tubular cells. Conclusions. L-cit supplementation established an anti-inflammatory profile and significantly preserved the nephron function during type 1

  9. Edaravone, a free radical scavenger, protects liver against valproic acid induced toxicity

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    Cakmak Neziha Hacihasanoglu

    2015-01-01

    Full Text Available Valproic acid (VPA, is a well established anticonvulsant drug that has been increasingly used in the treatment of many forms of generalized epilepsy. Edaravone (EDA; 3-methyl-1-phenyl-2-pyrazoline-5-one is a potent free radical scavenger. In this study, we aimed to investigate the effects of EDA on VPA-induced hepatic damage. Male Sprague Dawley rats were divided into four groups. Group I was control animals. Group II was control rats given valproic acid (500 mg kg-1 day for seven days. Group III was given only EDA (30 mg kg-1day for seven days. Group IV was given VPA+EDA (in same dose and time. EDA and VPA were given intraperitoneally. On the 8th day of experiment, blood samples and liver tissue were taken. Serum aspartate and alanine aminotransferase, alkaline phosphatase and bilirubin levels, liver myeloperoxidase, xanthine oxidase, adenosine deaminase, Na+/K+ATPase, sorbitol dehydrogenase, glutamate dehydrogenase, DT-diaphorase, arginase and thromboplastic activities, lipid peroxidation, protein carbonyl levels were increased whereas paraoxonase, biotinidase activities and glutathione levels were decreased in VPA group. Application of EDA with VPA protected against VPA-induced effects. These results demonstrated that administration of EDA is a potentially beneficial agent to reduce hepatic damage in VPA induced hepatotoxicity, probably by decreasing oxidative stress.

  10. The Immune System as a New Possible Cell Target for AFP 464 in a Spontaneous Mammary Cancer Mouse Model.

    Science.gov (United States)

    Callero, Mariana A; Rodriguez, Cristina E; Sólimo, Aldana; Bal de Kier Joffé, Elisa; Loaiza Perez, Andrea I

    2017-02-16

    Aminoflavone (AFP 464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER +). AFP 464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor size and growth rate in the estrogen dependent, Tamoxifen-sensitive spontaneous M05 mouse model. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, we investigated whether AFP 464 modulates the immune response in our mouse model. Studies on the effect of AFP 464 on the immune system were carried in BALB/c mice bearing M05 semi-differentiated mammary adenocarcinomas that express estrogen and progesterone receptors. Splenic cells and tumor inflammatory infiltrates were studied by cytometric analyses. The modulation of splenocytes cytotoxic activity by AFP 464 was also evaluated. We further investigated the effects of AFP 464 on peritoneal macrophages by evaluating metalloproteinase, arginase and iNOS activities. We found that AFP 464 increased splenic cytotoxic activity, diminished the number of systemic and local Treg lymphocytes and MDSCs, and induced a M1 phenotype in peritoneal macrophages of M05 tumor bearing mice. Therefore, we conclude that AFP 464 modulates immune response which collaborates with its anti-tumor activity. Our results place the immune system as a novel target for this anti-cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. This article is protected by copyright. All rights reserved.

  11. Myeloid-derived suppressor cells mediate tolerance induction in autoimmune disease.

    Science.gov (United States)

    Wegner, Anja; Verhagen, Johan; Wraith, David C

    2017-01-31

    In multiple sclerosis (MS) T cells aberrantly recognize self-peptides of the myelin sheath and attack the central nervous system (CNS). Antigen-specific peptide immunotherapy, which aims to restore tolerance while avoiding the use of non-specific immunosuppressive drugs, is a promising approach to combat autoimmune disease, but the cellular mechanisms behind successful therapy remain poorly understood. Myeloid-derived suppressor cells (MDSCs) have been studied intensively in the field of cancer and to a lesser extent in autoimmunity. Because of their suppressive effect on the immune system in cancer, we hypothesized that the development of MDSCs and their interaction with CD4(+) T cells could be beneficial for antigen-specific immunotherapy. Hence, changes in the quantity, phenotype and function of MDSCs during tolerance induction in our model of MS were evaluated. We reveal, for the first time, an involvement of a subset of MDSCs, known as polymorphonuclear (PMN)-MDSCs, in the process of tolerance induction. PMN-MDSCs were shown to adopt a more suppressive phenotype during peptide immunotherapy and inhibit CD4(+) T-cell proliferation in a cell-contact-dependent manner, mediated by arginase-1. Moreover, increased numbers of tolerogenic PMN-MDSCs, such as observed over the course of peptide immunotherapy, were demonstrated to provide protection from disease in a model of experimental autoimmune encephalomyelitis.

  12. Rationale and design of a proof-of-concept trial investigating the effect of uninterrupted perioperative (parenteral nutrition on amino acid profile, cardiomyocytes structure, and cardiac perfusion and metabolism of patients undergoing coronary artery bypass grafting

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    Cocchieri Riccardo

    2011-03-01

    Full Text Available Abstract Background Malnutrition is very common in patients undergoing cardiac surgery. Malnutrition can change myocardial substrate utilization which can induce adverse effects on myocardial metabolism and function. We aim to investigate the hypothesis that there is a disturbed amino acids profile in the cardiac surgical patient which can be normalized by (parenteral nutrition before, during and after surgery, subsequently improving cardiomyocyte structure, cardiac perfusion and glucose metabolism. Methods/Design This randomized controlled intervention study investigates the effect of uninterrupted perioperative (parenteral nutrition on cardiac function in 48 patients undergoing coronary artery bypass grafting. Patients are given enteral nutrition (n = 16 or parenteral nutrition (n = 16, at least two days before, during, and two days after coronary artery bypass grafting, or are treated according to the standard guidelines (control (n = 16. We will illustrate the effect of (parenteral nutrition on differences in concentrations of amino acids and asymmetric dimethylarginine and in activity of dimethylarginine dimethylaminohydrolase and arginase in cardiac tissue and blood plasma. In addition, cardiomyocyte structure by histological, immuno-histochemical and ultrastructural analysis will be compared between the (parenteral and control group. Furthermore, differences in cardiac perfusion and global left ventricular function and glucose metabolism, and their changes after coronary artery bypass grafting are evaluated by electrocardiography-gated myocardial perfusion scintigraphy and 18F-fluorodeoxy-glucose positron emission tomography respectively. Finally, fat free mass is measured before and after intervention with bioelectrical impedance spectrometry in order to evaluate nutritional status. Trial registration Netherlands Trial Register (NTR: NTR2183

  13. The farnesoid-X-receptor in myeloid cells controls CNS autoimmunity in an IL-10-dependent fashion.

    Science.gov (United States)

    Hucke, Stephanie; Herold, Martin; Liebmann, Marie; Freise, Nicole; Lindner, Maren; Fleck, Ann-Katrin; Zenker, Stefanie; Thiebes, Stephanie; Fernandez-Orth, Juncal; Buck, Dorothea; Luessi, Felix; Meuth, Sven G; Zipp, Frauke; Hemmer, Bernhard; Engel, Daniel Robert; Roth, Johannes; Kuhlmann, Tanja; Wiendl, Heinz; Klotz, Luisa

    2016-09-01

    Innate immune responses by myeloid cells decisively contribute to perpetuation of central nervous system (CNS) autoimmunity and their pharmacologic modulation represents a promising strategy to prevent disease progression in Multiple Sclerosis (MS). Based on our observation that peripheral immune cells from relapsing-remitting and primary progressive MS patients exhibited strongly decreased levels of the bile acid receptor FXR (farnesoid-X-receptor, NR1H4), we evaluated its potential relevance as therapeutic target for control of established CNS autoimmunity. Pharmacological FXR activation promoted generation of anti-inflammatory macrophages characterized by arginase-1, increased IL-10 production, and suppression of T cell responses. In mice, FXR activation ameliorated CNS autoimmunity in an IL-10-dependent fashion and even suppressed advanced clinical disease upon therapeutic administration. In analogy to rodents, pharmacological FXR activation in human monocytes from healthy controls and MS patients induced an anti-inflammatory phenotype with suppressive properties including control of effector T cell proliferation. We therefore, propose an important role of FXR in control of T cell-mediated autoimmunity by promoting anti-inflammatory macrophage responses.

  14. The role of arginine in infection and sepsis.

    Science.gov (United States)

    Luiking, Yvette C; Poeze, Martijn; Ramsay, Graham; Deutz, Nicolaas E P

    2005-01-01

    Sepsis is a systemic response to an infection, with high morbidity and mortality rates. Metabolic changes during infection and sepsis could be related to changes in metabolism of the amino acid L-arginine. In sepsis, protein breakdown is increased, which is a key process to maintain arginine delivery because both endogenous de novo arginine production from citrulline and food intake are reduced. Arginine catabolism, on the other hand, is markedly increased by enhanced use of arginine via the arginase and nitric oxide pathways. As a result, lowered plasma arginine levels are usually found. Arginine may therefore be considered as an essential amino acid in sepsis, and supplementation could be beneficial in sepsis by improving microcirculation and protein anabolism. L-Arginine supplementation in a hyperdynamic pig model of sepsis prohibits the increase in pulmonary arterial blood pressure, improves muscle and liver protein metabolism, and restores the intestinal motility pattern. Arguments raised against arginine supplementation are mainly pointed at stimulating nitric oxide (NO) production, with concerns about toxicity of increased NO and hemodynamic instability with refractory hypotension. NO synthase inhibition, however, increased mortality. Arginine supplementation in septic patients has transient effects on hemodynamics when supplied as a bolus but seems without hemodynamic side effects when supplied continuously. In conclusion, arginine could have an essential role in infection and sepsis.

  15. Exosomes released by granulocytic myeloid-derived suppressor cells attenuate DSS-induced colitis in mice.

    Science.gov (United States)

    Wang, Yungang; Tian, Jie; Tang, Xinyi; Rui, Ke; Tian, Xinyu; Ma, Jie; Ma, Bin; Xu, Huaxi; Lu, Liwei; Wang, Shengjun

    2016-03-29

    Myeloid-derived suppressor cells (MDSC) have been described in inflammatory bowel disease (IBD), but their role in the disease remains controversial. We sought to define the effect of granulocytic MDSC-derived exosomes (G-MDSC exo) in dextran sulphate sodium (DSS)-induced murine colitis. G-MDSC exo-treated mice showed greater resistance to colitis, as reflected by lower disease activity index, decreased inflammatory cell infiltration damage. There was a decrease in the proportion of Th1 cells and an increase in the proportion of regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) from G-MDSC exo-treated colitis mice. Moreover, lower serum levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected in G-MDSC exo-treated colitis mice. Interestingly, inhibition of arginase (Arg)-1 activity in G-MDSC exo partially abrogated the spontaneous improvement of colitis. In addition, G-MDSC exo could suppress CD4+ T cell proliferation and IFN-γ secretion in vitro and inhibit the delayed-type hypersensitivity (DTH) response, and these abilities were associated with Arg-1 activity. Moreover, G-MDSC exo promoted the expansion of Tregs in vitro. Taken together, these results suggest that G-MDSC exo attenuate DSS-induced colitis through inhibiting Th1 cells proliferation and promoting Tregs expansion.

  16. Extraintestinal Helminth Infection Limits Pathology and Proinflammatory Cytokine Expression during DSS-Induced Ulcerative Colitis: A Role for Alternatively Activated Macrophages and Prostaglandins.

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    Ledesma-Soto, Yadira; Callejas, Blanca E; Terrazas, César A; Reyes, Jose L; Espinoza-Jiménez, Arlett; González, Marisol I; León-Cabrera, Sonia; Morales, Rosario; Olguín, Jonadab E; Saavedra, Rafael; Oghumu, Steve; Satoskar, Abhay R; Terrazas, Luis I

    2015-01-01

    Chronic inflammation of the intestinal mucosa is characteristic of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Helminth parasites have developed immunomodulatory strategies that may impact the outcome of several inflammatory diseases. Therefore, we investigated whether Taenia crassiceps infection is able to decrease the inflammatory effects of dextran sulfate sodium- (DSS-) induced ulcerative colitis in BALB/c and C57BL/6 mice. Preinfection significantly reduced the manifestations of DSS-induced colitis, as weight loss and shortened colon length, and decreased the disease activity index independently of the genetic background of the mice. Taenia infection decreased systemic levels of proinflammatory cytokines while increasing levels of IL-4 and IL-10, and the inflammatory infiltrate into the colon was also markedly reduced. RT-PCR assays from colon showed that T. crassiceps-infected mice displayed increased expression of Arginase-1 but decreased expression of iNOS compared to DSS-treated uninfected mice. The percentages of T regulatory cells were not increased. The adoptive transfer of alternatively activated macrophages (AAMФs) from infected mice into mice with DSS-induced colitis reduced the severity of colon inflammation. Administration of indomethacin abrogated the anticolitic effect of Taenia. Thus, T. crassiceps infection limits the pathology of ulcerative colitis by suppressing inflammatory responses mechanistically associated with AAMФs and prostaglandins.

  17. Improving long term outcomes in urea cycle disorders-report from the Urea Cycle Disorders Consortium.

    Science.gov (United States)

    Waisbren, Susan E; Gropman, Andrea L; Batshaw, Mark L

    2016-07-01

    The Urea Cycle Disorders Consortium (UCDC) has conducted, beginning in 2006, a longitudinal study (LS) of eight enzyme deficiencies/transporter defects associated with the urea cycle. These include N-acetylglutamate synthase deficiency (NAGSD); Carbamyl phosphate synthetase 1 deficiency (CPS1D); Ornithine transcarbamylase deficiency (OTCD); Argininosuccinate synthetase deficiency (ASSD) (Citrullinemia); Argininosuccinate lyase deficiency (ASLD) (Argininosuccinic aciduria); Arginase deficiency (ARGD, Argininemia); Hyperornithinemia, hyperammonemia, homocitrullinuria (HHH) syndrome (or mitochondrial ornithine transporter 1 deficiency [ORNT1D]); and Citrullinemia type II (mitochondrial aspartate/glutamate carrier deficiency [CITRIN]). There were 678 UCD patients enrolled in 14 sites in the U.S., Canada, and Europe at the writing of this paper. This review summarizes findings of the consortium related to outcome, focusing primarily on neuroimaging findings and neurocognitive function. Neuroimaging studies in late onset OTCD offered evidence that brain injury caused by biochemical dysregulation may impact functional neuroanatomy serving working memory processes, an important component of executive function and regulation. Additionally, there were alteration in white mater microstructure and functional connectivity at rest. Intellectual deficits in OTCD and other urea cycle disorders (UCD) vary. However, when neuropsychological deficits occur, they tend to be more prominent in motor/performance areas on both intelligence tests and other measures. In some disorders, adults performed significantly less well than younger patients. Further longitudinal follow-up will reveal whether this is due to declines throughout life or to improvements in diagnostics (especially newborn screening) and treatments in the younger generation of patients.

  18. In Vitro and In Vivo Anticancer Effects of Sterol Fraction from Red Algae Porphyra dentata

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    Katarzyna Kazłowska

    2013-01-01

    Full Text Available Porphyra dentata, an edible red macroalgae, is used as a folk medicine in Asia. This study evaluated in vitro and in vivo the protective effect of a sterol fraction from P. dentata against breast cancer linked to tumor-induced myeloid derived-suppressor cells (MDSCs. A sterol fraction containing cholesterol, β-sitosterol, and campesterol was prepared by solvent fractionation of methanol extract of P. dentata  in silica gel column chromatography. This sterol fraction in vitro significantly inhibited cell growth and induced apoptosis in 4T1 cancer cells. Intraperitoneal injection of this sterol fraction at 10 and 25 mg/kg body weight into 4T1 cell-implanted tumor BALB/c mice significantly inhibited the growth of tumor nodules and increased the survival rate of mice. This sterol fraction significantly decreased the reactive oxygen species (ROS and arginase activity of MDSCs in tumor-bearing mice. Therefore, the sterol fraction from P. dentata showed potential for protecting an organism from 4T1 cell-based tumor genesis.

  19. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway

    Science.gov (United States)

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-01

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by Nω-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension. PMID:26739766

  20. Investigating the Synergistic Effects of Combined Modified Alginates on Macrophage Phenotype

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    Hannah C. Bygd

    2016-12-01

    Full Text Available Understanding macrophage responses to biomaterials is crucial to the success of implanted medical devices, tissue engineering scaffolds, and drug delivery vehicles. Cellular responses to materials may depend synergistically on multiple surface chemistries, due to the polyvalent nature of cell–ligand interactions. Previous work in our lab found that different surface functionalities of chemically modified alginate could sway macrophage phenotype toward either the pro-inflammatory or pro-angiogenic phenotype. Using these findings, this research aims to understand the relationship between combined material surface chemistries and macrophage phenotype. Tumor necrosis factor-α (TNF-α secretion, nitrite production, and arginase activity were measured and used to determine the ability of the materials to alter macrophage phenotype. Cooperative relationships between pairwise modifications of alginate were determined by calculating synergy values for the aforementioned molecules. Several materials appeared to improve M1 to M2 macrophage reprogramming capabilities, giving valuable insight into the complexity of surface chemistries needed for optimal incorporation and survival of implanted biomaterials.

  1. The synthetic melanocortin (CKPV2 exerts anti-fungal and anti-inflammatory effects against Candida albicans vaginitis via inducing macrophage M2 polarization.

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    Hai-xia Ji

    Full Text Available In this study, we examined anti-fungal and anti-inflammatory effects of the synthetic melanocortin peptide (Ac-Cys-Lys-Pro-Val-NH22 or (CKPV2 against Candida albicans vaginitis. Our in vitro results showed that (CKPV2 dose-dependently inhibited Candida albicans colonies formation. In a rat Candida albicans vaginitis model, (CKPV2 significantly inhibited vaginal Candida albicans survival and macrophages sub-epithelial mucosa infiltration. For mechanisms study, we observed that (CKPV2 inhibited macrophages phagocytosis of Candida albicans. Meanwhile, (CKPV2 administration inhibited macrophage pro-inflammatory cytokines (TNF-α, IL-1β and IL-6 release, while increasing the arginase activity and anti-inflammatory cytokine IL-10 production, suggesting macrophages M1 to M2 polarization. Cyclic AMP (cAMP production was also induced by (CKPV2 administration in macrophages. These above effects on macrophages by (CKPV2 were almost reversed by melanocortin receptor-1(MC1R siRNA knockdown, indicating the requirement of MC1R in the process. Altogether, our results suggest that (CKPV2 exerted anti-fungal and anti-inflammatory activities against Candida albicans vaginitis probably through inducing macrophages M1 to M2 polarization and MC1R activation.

  2. Extracellular mycobacterial DnaK polarizes macrophages to the M2-like phenotype.

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    Rafael L Lopes

    Full Text Available Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses.

  3. IL-10 and ARG-1 Concentrations in Bone Marrow and Peripheral Blood of Metastatic Neuroblastoma Patients Do Not Associate with Clinical Outcome

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    Fabio Morandi

    2015-01-01

    Full Text Available The expression of the immunosuppressive molecules IL-10 and arginase 1 (ARG-1, and of FOXP3 and CD163, as markers of regulatory T cells (Treg and macrophages, respectively, was evaluated in bone marrow (BM and peripheral blood (PB samples collected at diagnosis from patients with metastatic neuroblastoma (NB. IL-10 and ARG-1 plasma concentrations were measured and the association of each parameter with patients’ outcome was tested. The percentages of immunosuppressive Treg and type-1 regulatory (Tr1 cells were also determined. In both BM and PB samples, IL-10 mRNA expression was higher in metastatic NB patients than in controls. IL-10 plasma concentration was higher in patients with NB regardless of stage. Neither IL-10 expression nor IL-10 plasma concentration significantly associated with patient survival. In PB samples from metastatic NB patients, ARG-1 and CD163 expression was higher than in controls but their expression did not associate with survival. Moreover, ARG-1 plasma concentration was lower than in controls, and no association with patient outcome was found. Finally, in metastatic NB patients, the percentage of circulating Treg was higher than in controls, whereas that of Tr1 cells was lower. In conclusion, although IL-10 concentration and Treg percentage were increased, their contribution to the natural history of metastatic NB appears uncertain.

  4. Integration of nondegradable polystyrene and degradable gelatin in a core-sheath nanofibrous patch for pelvic reconstruction.

    Science.gov (United States)

    Ge, Liangpeng; Li, Qingtao; Jiang, Junzi; You, Xiaoyan; Liu, Zuohua; Zhong, Wen; Huang, Yong; Xing, Malcolm M Q

    2015-01-01

    Pelvic organ prolapse (POP) is a serious health issue affecting many adult women. Complications of POP include pelvic pressure, pelvic pain, and problems in emptying their bowels or bladder. Sometimes, POP may even cause urinary outflow obstruction and lead to bladder or kidney infections. Currently, synthetic and naturally derived materials have been chosen for treatment of POP to reduce the high recurrence rates after surgical interventions. However, existing materials for POP treatment cannot meet the clinical requirements in terms of biocompatibility, mechanics, and minimal risk of rejection. Especially, erosion in synthetic polymers and rapid degradation in natural polymers limit their further applications in clinics. To address these concerns, we report a novel POP replacement using core-sheath polystyrene/gelatin electrospun nanofiber mesh. The outside gelatin sheath provides a hydrophilic surface and implantable integrity between host and guest, while the inner PS core offers the necessary mechanical support. The composite mesh shows graft accommodation in pelvic submucosa after implantation in vivo, as shown in hematoxylin-eosin staining and T helper cell phenotype and macrophage phenotype stainings. Qualitative analysis of inducible nitric oxide synthase, arginase, interferon-γ, and interleukin-10 gene expressions also indicates that the implanted composite mesh switches to accommodation mode 2 weeks postimplantation. Thus, these novel core-sheath polystyrene/gelatin nanofibrous membranes are promising in pelvic reconstruction.

  5. Butylated Hydroxyanisole Blocks the Occurrence of Tumor Associated Macrophages in Tobacco Smoke Carcinogen-Induced Lung Tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yan; Choksi, Swati; Liu, Zheng-Gang, E-mail: zgliu@helix.nih.gov [Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-12-04

    Tumor-associated macrophages (TAMs) promote tumorigenesis because of their proangiogenic and immune-suppressive functions. Here, we report that butylated hydroxyanisole (BHA) blocks occurrence of tumor associated macrophages (TAMs) in tobacco smoke carcinogen-induced lung tumorigenesis. Continuous administration of butylated hydroxyanisole (BHA), a ROS inhibitor, before or after NNK treatment significantly blocked tumor development, although less effectively when BHA is administered after NNK treatment. Strikingly, BHA abolished the occurrence of F4/80{sup +} macrophages with similar efficiency no matter whether it was administered before or after NNK treatment. Detection of cells from bronchioalveolar lavage fluid (BALF) confirmed that BHA markedly inhibited the accumulation of macrophages while slightly reducing the number of lymphocytes that were induced by NNK. Immunohistological staining showed that BHA specifically abolished the occurrence of CD206{sup +} TAMs when it was administered before or after NNK treatment. Western blot analysis of TAMs markers, arginase I and Ym-1, showed that BHA blocked NNK-induced TAMs accumulation. Our study clearly demonstrated that inhibiting the occurrence of TAMs by BHA contributes to the inhibition of tobacco smoke carcinogen-induced tumorigenesis, suggesting ROS inhibitors may serve as a therapeutic target for treating smoke-induced lung cancer.

  6. Gypenoside Attenuates β Amyloid-Induced Inflammation in N9 Microglial Cells via SOCS1 Signaling

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    Hui Cai

    2016-01-01

    Full Text Available Reducing β amyloid- (Aβ- induced microglial activation is believed to be effective in treating Alzheimer’s disease (AD. Microglia can be activated into classic activated state (M1 state or alternative activated state (M2 state, and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1. In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α, interleukin 1β (IL-1β, and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1 expression, IL-10, brain-derived neurotrophic factor (BDNF, and glial cell-derived neurotrophic factor (GDNF releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1.

  7. Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism.

    Science.gov (United States)

    Weinstein, L H; Osmeloski, J F; Wettlaufer, S H; Galston, A W

    1987-01-01

    In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.

  8. In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues.

    Science.gov (United States)

    Slocum, R D; Galston, A W

    1985-01-01

    Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.

  9. The effects of some polyamine biosynthetic inhibitors on growth and morphology of phytopathogenic fungi.

    Science.gov (United States)

    Rajam, M V; Galston, A W

    1985-01-01

    We have studied the effects of two polyamine biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA), and of polyamines (PAs), alone and in combination, on mycelial growth and morphology of four phytopathogenic fungi: Botrytis sp, B. cinerea, Rhizoctonia solani and Monilinia fructicola. The inhibitors were added to a Czapek agar medium to get final concentrations of 0.1, 0.5 and 1.0 mM. DFMO and DFMA, suicide inhibitors of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) respectively, inhibited mycelial growth strongly; the effect was generally more pronounced with DFMA than with DFMO, but each fungus had its own response pattern. The addition of the PAs putrescine (Put) and spermidine (Spd) to the culture medium resulted in a promotion of growth. In Botrytis sp and Monilinia fructicola exposed to inhibitors plus PAs, mycelial growth was actually increased above control values. Mycelial morphology was altered and cell size dramatically reduced in plates containing inhibitors alone, whereas with PAs alone, or in combination with inhibitors, morphology was normal, but cell length and diameters increased considerably. These results suggest that PAs are essential for growth in fungal mycelia. The inhibition caused by DFMA may be due to its arginase-mediated conversion to DFMO.

  10. The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum.

    Science.gov (United States)

    Pieckenstain, F L; Gárriz, A; Chornomaz, E M; Sánchez, D H; Ruiz, O A

    2001-12-01

    We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. Alpha-Difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.

  11. Asthma in Sickle Cell Disease

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    Manisha Newaskar

    2011-01-01

    Full Text Available In recent years, evidence has increased that asthma predisposes to complications of sickle cell disease (SCD, such as pain crises, acute chest syndrome, pulmonary hypertension, and stroke, and is associated with increased mortality. An obstructive pattern of pulmonary function, along with a higher-than-expected prevalence of airway hyper-responsiveness (AHR when compared to the general population, has led some researchers to suspect that underlying hemolysis may contribute to the development of a pulmonary disease similar to asthma in patients with SCD. While the pathophysiologic mechanism in atopic asthma involves up-regulation of Th2 cytokines, mast cell– and eosinophil-driven inflammation, plus increased activity of inducible nitric oxide synthase (iNOS and arginase in airway epithelium resulting in obstructive changes and AHR, the exact mechanisms of AHR, obstructive and restrictive lung disease in SCD is unclear. It is known that SCD is associated with a proinflammatory state and an enhanced inflammatory response is seen during vaso-occlusive events (VOE. Hemolysis-driven acute-on-chronic inflammation and dysregulated arginines–nitric oxide metabolism are potential mechanisms by which pulmonary dysfunction could occur in patients with SCD. In patients with a genetic predisposition of atopic asthma, these changes are probably more severe and result in increased susceptibility to sickle cell complications. Early recognition and aggressive management of asthma based on established National Institutes of Health asthma guidelines is recommended in order to minimize morbidity and mortality.

  12. The Macrophage Galactose-Type Lectin-1 (MGL1 Recognizes Taenia crassiceps Antigens, Triggers Intracellular Signaling, and Is Critical for Resistance to This Infection

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    Daniel Montero-Barrera

    2015-01-01

    Full Text Available C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1 recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1−/− mice showed less binding ability toward parasite antigens than their wild-type (WT counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1−/− macrophages. Following T. crassiceps infection, MGL1−/− mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1−/− mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1−/− macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance.

  13. Gr-1+CD11b+ cells are responsible for tumor promoting effect of TGF-β in breast cancer progression.

    Science.gov (United States)

    Li, Zhaoyang; Pang, Yanli; Gara, Sudheer Kumar; Achyut, B R; Heger, Christopher; Goldsmith, Paul K; Lonning, Scott; Yang, Li

    2012-12-01

    One great challenge in our understanding of TGF-β cancer biology and the successful application of TGF-β-targeted therapy is that TGF-β works as both a tumor suppressor and a tumor promoter. The underlying mechanisms for its functional change remain to be elucidated. Using 4T1 mammary tumor model that shares many characteristics with human breast cancer, particularly its ability to spontaneously metastasize to the lungs, we demonstrate that Gr-1+CD11b+ cells or myeloid derived suppressor cells are important mediators in TGF-β regulation of mammary tumor progression. Depletion of Gr-1+CD11b+ cells diminished the antitumor effect of TGF-β neutralization. Two mechanisms were involved: first, treatment with TGF-β neutralization antibody (1D11) significantly decreased the number of Gr-1+CD11b+ cells in tumor tissues and premetastatic lung. This is mediated through increased Gr-1+CD11b+ cell apoptosis. In addition, 1D11 treatment significantly decreased the expression of Th2 cytokines and Arginase 1. Interestingly, the number and property of Gr-1+CD11b+ cells in peripheral blood/draining lymph nodes correlated with tumor size and metastases in response to 1D11 treatment. Our data suggest that the efficacy of TGF-β neutralization depends on the presence of Gr-1+CD11b+ cells, and these cells could be good biomarkers for TGF-β-targeted therapy.

  14. Effect of L-arginine on metabolism of polyamines in rat's brain with extrahepatic cholestasis.

    Science.gov (United States)

    Sokolovic, Dusan; Bjelakovic, Gordana; Nikolic, Jelenka; Djindjic, Boris; Pavlovic, Dusica; Kocic, Gordana; Stojanovic, Ivana; Pavlovic, Voja

    2010-01-01

    Cholestatic encephalopathy results from accumulation of unconjugated bilirubin and hydrophobic bile acids in the brain. The aim of this study was to determine disturbances of polyamine metabolism in the brains of rats with experimental extrahepatic cholestasis and the effects of L-arginine administration. Wister rats were divided into groups: I: sham-operated, II: rats treated with L-arginine, III: animals with bile-duct ligation (BDL), and IV: cholestatic-BDL rats treated with L-arginine. Increased plasma gamma-glutamyltransferase and alkaline phosphatase activity and increased bile-acids and bilirubin levels in BDL rats were reduced by administration of L-arginine (P < 0.001). Cholestasis increased the brain's putrescine (P < 0.001) and decreased spermidine and spermine concentration (P < 0.05). The activity of polyamine oxidase was increased (P < 0.001) and diamine oxidase was decreased (P < 0.001) in the brains of BDL rats. Cholestasis increased the activity of arginase (P < 0.05) and decreased the level of citrulline (P < 0.001). Administration of L-arginine in BDL rats prevents metabolic disorders of polyamines and establishes a neuroprotective role in the brain during cholestasis.

  15. Cocoa and human health.

    Science.gov (United States)

    Ellam, Samantha; Williamson, Gary

    2013-01-01

    Cocoa is a dry, powdered, nonfat component product prepared from the seeds of the Theobroma cacao L. tree and is a common ingredient of many food products, particularly chocolate. Nutritionally, cocoa contains biologically active substances that may affect human health: flavonoids (epicatechin and oligomeric procyanidins), theobromine, and magnesium. Theobromine and epicatechin are absorbed efficiently in the small intestine, and the nature of their conjugates and metabolites are now known. Oligomeric procyanidins are poorly absorbed in the small intestine, but catabolites are very efficiently absorbed after microbial biotransformation in the colon. A significant number of studies, using in vitro and in vivo approaches, on the effects of cocoa and its constituent flavonoids have been conducted. Most human intervention studies have been performed on cocoa as an ingredient, whereas many in vitro studies have been performed on individual components. Approximately 70 human intervention studies have been carried out on cocoa and cocoa-containing products over the past 12 years, with a variety of endpoints. These studies indicate that the most robust biomarkers affected are endothelial function, blood pressure, and cholesterol level. Mechanistically, supporting evidence shows that epicatechin affects nitric oxide synthesis and breakdown (via inhibition of nicotinamide adenine di-nucleotide phosphate oxidase) and the substrate arginine (via inhibition of arginase), among other targets. Evidence further supports cocoa as a biologically active ingredient with potential benefits on biomarkers related to cardiovascular disease. However, the calorie and sugar content of chocolate and its contribution to the total diet should be taken into account in intervention studies.

  16. Visceral leishmaniasis patients display altered composition and maturity of neutrophils as well as impaired neutrophil effector functions

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    Endalew Yizengaw

    2016-11-01

    Full Text Available Immunologically, active visceral leishmaniasis (VL is characterised by profound immunosuppression, severe systemic inflammatory responses and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis, however, their role in human visceral leishmaniasis is poorly understood.In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analysed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species and phagocytose bacterial particles, but not Leishmania parasites.Our results suggest that impaired effector functions, increased activation and immaturity of neutrophils play a key role in the pathogenesis of VL.

  17. TNF neutralization results in the delay of transplantable tumor growth and reduced MDSC accumulation.

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    Kamar-Sulu N. Atretkhany

    2016-04-01

    Full Text Available Myeloid-derived suppressor cells (MDSC represent a heterogeneous population of immature myeloid cells that under normal conditions may differentiate into mature macrophages, granulocytes and dendritic cells. However, under pathological conditions associated with inflammation, cancer or infection such differentiation is inhibited leading to immature myeloid cell expansion. Under the influence of inflammatory cytokines, these cells become MDSC, acquire immunosuppressive phenotype and accumulate in the affected tissue, as well as in the periphery. Immune suppressive activity of MDSC is partly due to upregulation of arginase 1 (Arg1, inducible nitric oxide synthase (iNOS and anti-inflammatory cytokines, such as IL-10 and TGF-β. These suppressive factors can enhance tumor growth by repressing T-cell-mediated anti-tumor responses. TNF is a critical factor for induction, expansion and suppressive activity of MDSC. In this study we evaluated the effects of systemic TNF ablation on tumor-induced expansion of MDSC in vivo using TNF humanized (hTNF KI mice. Both Etanercept and Infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume and in less accumulated MDSC in the blood three weeks after tumor cell inoculation. Thus, our study uncovers anti-tumor effects of systemic TNF ablation in vivo.

  18. TISSUE FACTOR EXPRESSION BY MYELOID CELLS CONTRIBUTES TO PROTECTIVE IMMUNE RESPONSE AGAINST Mycobacterium tuberculosis INFECTION

    Science.gov (United States)

    Venkatasubramanian, Sambasivan; Tripathi, Deepak; Tucker, Torry; Paidipally, Padmaja; Cheekatla, Satyanarayana; Welch, Elwyn; Raghunath, Anjana; Jeffers, Ann; Tvinnereim, Amy R.; Schechter, Melissa E; Andrade, Bruno B; Mackman, Nizel; Idell, Steven; Vankayalapati, Ramakrishna

    2015-01-01

    Tissue Factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TFΔ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2 like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth. PMID:26471500

  19. Monocyte Differentiation towards Protumor Activity Does Not Correlate with M1 or M2 Phenotypes

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    G. Karina Chimal-Ramírez

    2016-01-01

    Full Text Available Macrophages facilitate breast cancer progression. Macrophages were initially classified as M1 or M2 based on their distinct metabolic programs and then expanded to include antitumoral (M1 and protumoral (M2 activities. However, it is still uncertain what markers define the pro- and antitumoral phenotypes and what conditions lead to their formation. In this study, monocytic cell lines and primary monocytes were subjected to commonly reported protocols of M1/M2 polarization and conditions known to engage monocytes into protumoral functions. The results showed that only IDO enzyme and CD86 M1 markers were upregulated correlating with M1 polarization. TNF-α, CCR7, IL-10, arginase I, CD36, and CD163 were expressed indistinguishably from M1 or M2 polarization. Similarly, protumoral engaging resulted in upregulation of both M1 and M2 markers, with conditioned media from the most aggressive breast cancer cell line promoting the greatest changes. In spite of the mixed phenotype, M1-polarized macrophages exhibited the highest expression/secretion of inflammatory mediators, many of which have previously been associated with breast cancer aggressiveness. These data argue that although the existence of protumoral macrophages is unquestionable, their associated phenotypes and the precise conditions driving their formation are still unclear, and those conditions may need both M1 and M2 stimuli.

  20. A preliminary study to understand the effect of Fasciola hepatica tegument on naïve macrophages and humoral responses in an ovine model.

    Science.gov (United States)

    Haçarız, O; Sayers, G; Mulcahy, G

    2011-02-15

    Fasciola hepatica, the liver fluke, is a highly evolved endo-parasite that uses various mechanisms to evade the host immune system. The immunosuppressive capabilities of the parasite's excretory/secretory products have been well demonstrated by previous independent studies. However, the role of the parasite's tegument in the immune responses remains to be investigated. In this study, the effect of the tegument and other fractions of adult F. hepatica (excretory/secretory, liver fluke homogenate and liver fluke homogenate without tegument) in the activation of naïve macrophages in vitro was investigated using an ovine model. In addition, an immunoproteomic approach was used to investigate the characteristics of humoral antibody responses developed in sheep against the tegument fraction. The results indicated significantly increased arginase expression in macrophages incubated with the tegument and excretory/secretory fractions. Two dimensional gel electrophoresis of the tegument demonstrated approximately 100 protein spots, with only four of these spots were highly reactive with the positive serum as determined by 2-DE immunoblotting. These results give a preliminary indication that the liver fluke tegument may play role in avoiding hosts' protective immune responses against itself.

  1. Activation of murine microglial N9 cells is attenuated through cannabinoid receptor CB2 signaling.

    Science.gov (United States)

    Ma, Lei; Jia, Ji; Liu, Xiangyu; Bai, Fuhai; Wang, Qiang; Xiong, Lize

    2015-02-27

    Inhibition of microglial activation is effective in treating various neurological disorders. Activation of microglial cannabinoid CB2 receptor induces anti-inflammatory effects, and the mechanism, however, is still elusive. Microglia could be activated into the classic activated state (M1 state) or the alternative activated state (M2 state), the former is cytotoxic, and the latter is neurotrophic. In this study, we used lipopolysaccharide (LPS) plus interferon-γ (IFNγ) to activate N9 microglia and hypothesized the pretreatment with cannabinoid CB2 receptor agonist AM1241 attenuates microglial activation by shifting microglial M1 to M2 state. We found that pretreatment with 5 μM AM1241 at 1 h before microglia were exposed to LPS plus IFNγ decreased the expression of inducible nitric oxide synthase (iNOS) and the release of pro-inflammatory factors, increased the expression of arginase 1 (Arg-1) and the release of anti-inflammatory and neurotrophic factors in microglia. However, these effects induced by AM1241 pretreatment were significantly reversed in the presence of 10 μM cannabinoid CB2 receptor antagonist AM630 or 10 μM protein kinase C (PKC) inhibitor chelerythrine. These findings indicated that AM1241 pretreatment attenuates microglial activation by shifting M1 to M2 activated state via CB2 receptor, and the AM1241-induced anti-inflammatory effects may be mediated by PKC.

  2. Role of orexin A signaling in dietary palmitic acid-activated microglial cells.

    Science.gov (United States)

    Duffy, Cayla M; Yuan, Ce; Wisdorf, Lauren E; Billington, Charles J; Kotz, Catherine M; Nixon, Joshua P; Butterick, Tammy A

    2015-10-08

    Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

  3. Chocolate: (un)healthy source of polyphenols?

    Science.gov (United States)

    Rimbach, Gerald; Egert, Sarah; de Pascual-Teresa, Sonia

    2011-02-01

    There is recent epidemiological evidence that chocolate consumption may improve vascular health. Furthermore, several small-scale human intervention studies indicate that habitual chocolate intake enhances the production of vasodilative nitric oxide and may lower blood pressure. It is hypothesized that potential beneficial effects of chocolate on vascular health are at least partly mediated by cocoa polyphenols including procyanidins. Based on cell culture studies, molecular targets of chocolate polyphenols are endothelial nitric oxide synthetase as well as arginase. However, human bioavailability studies suggest that the plasma concentrations of cocoa polyphenols are manifold lower than those concentrations used in cultured cells in vitro. The experimental evidence for beneficial vascular effects of chocolate in human interventions studies is yet not fully convincing. Some human intervention studies on chocolate and its polyphenols lack a stringent study design. They are sometimes underpowered and not always placebo controlled. Dietary chocolate intake in many of these human studies was up to 100 g per day. Since chocolate is a rich source of sugar and saturated fat, it is questionable whether chocolate could be recommended as part of a nutrition strategy to promote vascular health.

  4. Oxidative status imbalance in patients with metabolic syndrome: role of the myeloperoxidase/hydrogen peroxide axis.

    Science.gov (United States)

    da Fonseca, Lucas José Sá; Nunes-Souza, Valéria; Guedes, Glaucevane da Silva; Schettino-Silva, Glauber; Mota-Gomes, Marco Antônio; Rabelo, Luíza Antas

    2014-01-01

    The present study evaluated the cardiometabolic and redox balance profiles in patients with Metabolic Syndrome compared to apparently healthy individuals, and the participation of the myeloperoxidase/hydrogen peroxide axis in systemic lipid peroxidation. Twenty-four patients with Metabolic Syndrome and eighteen controls underwent a full clinical assessment. Venous blood samples were collected for general biochemical dosages, as well as for the oxidative stress analyses (superoxide dismutase, catalase, and arginase activities; and lipid peroxidation, myeloperoxidase activity, nitrite, and hydrogen peroxide concentrations in plasma). Arterial stiffness was assessed by radial artery applanation tonometry. Plasma lipid peroxidation, erythrocyte superoxide dismutase activity, myeloperoxidase activity, and hydrogen peroxide concentrations were shown to be increased in Metabolic Syndrome patients, without significant differences for the other enzymes, plasma nitrite concentrations, and arterial stiffness. Linear regression analysis revealed a positive and significant correlation between lipid peroxidation and myeloperoxidase and also between this enzyme and hydrogen peroxide. In contrast, such correlation was not observed between lipid peroxidation and hydrogen peroxide. In summary, Metabolic Syndrome patients exhibited evident systemic redox imbalance compared to controls, with the possible participation of the myeloperoxidase/hydrogen peroxide axis as a contributor in lipid peroxidation.

  5. L-arginine and L-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats.

    Science.gov (United States)

    El-Sheikh, Nora M; Khalil, Fatma A

    2011-04-01

    Sodium nitrite (NaNO(2)) is a flavoring, coloring and preservative agent in meat and fish products. The study aimed to evaluate the efficacy of L-arginine and L-glutamine supplementation as a potentially novel and useful strategy for the modulation of oxidative stress and toxicity induced by NaNO(2) in male rats. Rats were divided into six groups each of 10 rats and treated for 6 weeks: group 1 as normal control; group 2 fed standard diet containing 0.2% NaNO(2); group 3 and 4 fed the previous diet supplemented with 1% and 2% arginine, respectively; group 5 and 6 fed NaNO(2) diet supplemented with 1% and 2% glutamine, respectively. NaNO(2) treatment induced a significant increase in serum malondialdehyde, nitric oxide, arginase, glutathione-S-transferase activities, urea and creatinine as well as differential leucocytes%. However, a significant decrease was recorded in reduced glutathione, catalase activity, total protein, albumin and some hematological parameters as well as immunoglobulin G. On the other hand, arginine or glutamine showed a remarkable modulation of these abnormalities as indicated by reduction of malondialdehyde and improvement of the investigated antioxidant and hematological parameters. It can be concluded that arginine or glutamine supplementation may reduce oxidative stress and improve the hazard effects of NaNO(2).

  6. Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection.

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    Daniel P Barry

    Full Text Available Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2 is essential for transport of L-arginine (L-Arg into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO produced from inducible NO synthase (iNOS, or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/- mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/- mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/- mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.

  7. Macrophage reprogramming: influence of latex beads with various functional groups on macrophage phenotype and phagocytic uptake in vitro.

    Science.gov (United States)

    Akilbekova, Dana; Philiph, Rachel; Graham, Austin; Bratlie, Kaitlin M

    2015-01-01

    Macrophages play a crucial role in initiating immune responses with various functions ranging from wound healing to antimicrobial actions. The type of biomaterial is suggested to influence macrophage phenotype. Here, we show that exposing M1- and M2-activated macrophages to polystyrene latex beads bearing different functional groups can alter secretion profiles, providing a possible method for altering the course of the host response. Macrophages were stimulated with either lipopolysaccharide or interleukin (IL) 4 and cultured for 24 h with 10 different latex beads. Proinflammatory cytokines (tumor necrosis factor α, monocyte chemotactic protein 1) and nitrite served as markers for the M1 phenotype and proangiogenic cytokine (IL-10) and arginase activity for M2 cells. The ability of the macrophages to phagocytize Escherichia coli particles and water contact angles of the polymers were also assessed. Different patterns of cytokine expression and phagocytosis activity were induced by the various particles. Particles did not polarize the cells toward one specific phenotype versus another, but rather induced changes in both pro- and anti-inflammatory markers. Our results suggest a dependence of pro- and anti-inflammatory cytokines and phagocytic activities on material type and cytokine stimuli. These data also illustrate how biomaterials can be exploited to alter host responses for drug delivery and tissue engineering applications.

  8. Identification of stathmin 1 during peri-implantation period in mouse endometrium by a proteomics-based analysis.

    Science.gov (United States)

    Gou, Jinhai; Jia, Jia; Zhao, Xia; Yi, Tao; Li, Zhengyu

    2015-05-29

    In this work we aimed to identify the differentially expressed proteins and their potential roles during peri-implantation period through proteomics-based approach. Adult healthy female mice were mated naturally with fertile males to produce pregnancy. The models of pseudopregnancy, delayed implantation, and artificial decidualization were established. The protein profile between pre-implantation (D1) and implantation (D5) period was compared by two-dimensional electrophoresis (2-DE) and identified by mass spectrometry (MS). 2-DE yielded comparative images presenting over 500 protein spots in D1 and D5 mouse endometrium. 15 proteins were identified, of which stathmin 1, Apo-A1, hnRNP H3, transgelin 2 and arginase 1 were validated by western blotting. Stathmin 1 expression did not change in pseudopregnancy, but activation of implantation, or induction of decidualization increased it dramatically. Under non-pregnant status, progesterone alone or in combination with17β-estradiol increased it dramatically. Our results clarified the protein profile in mouse endometrium during implantation. The specific expression profile of stathmin 1 suggested that it should be involved in implantation and serve as a potential regulator of this process. These findings may contribute to the better understanding of the molecules events during embryo implantation, and subsequently improve the ability to treat infertility.

  9. Integration of nondegradable polystyrene and degradable gelatin in a core–sheath nanofibrous patch for pelvic reconstruction

    Science.gov (United States)

    Ge, Liangpeng; Li, Qingtao; Jiang, Junzi; You, Xiaoyan; Liu, Zuohua; Zhong, Wen; Huang, Yong; Xing, Malcolm MQ

    2015-01-01

    Pelvic organ prolapse (POP) is a serious health issue affecting many adult women. Complications of POP include pelvic pressure, pelvic pain, and problems in emptying their bowels or bladder. Sometimes, POP may even cause urinary outflow obstruction and lead to bladder or kidney infections. Currently, synthetic and naturally derived materials have been chosen for treatment of POP to reduce the high recurrence rates after surgical interventions. However, existing materials for POP treatment cannot meet the clinical requirements in terms of biocompatibility, mechanics, and minimal risk of rejection. Especially, erosion in synthetic polymers and rapid degradation in natural polymers limit their further applications in clinics. To address these concerns, we report a novel POP replacement using core–sheath polystyrene/gelatin electrospun nanofiber mesh. The outside gelatin sheath provides a hydrophilic surface and implantable integrity between host and guest, while the inner PS core offers the necessary mechanical support. The composite mesh shows graft accommodation in pelvic submucosa after implantation in vivo, as shown in hematoxylin–eosin staining and T helper cell phenotype and macrophage phenotype stainings. Qualitative analysis of inducible nitric oxide synthase, arginase, interferon-γ, and interleukin-10 gene expressions also indicates that the implanted composite mesh switches to accommodation mode 2 weeks postimplantation. Thus, these novel core–sheath polystyrene/gelatin nanofibrous membranes are promising in pelvic reconstruction. PMID:25995629

  10. Dynamic Changes of Microglia/Macrophage M1 and M2 Polarization in Theiler's Murine Encephalomyelitis.

    Science.gov (United States)

    Herder, Vanessa; Iskandar, Cut Dahlia; Kegler, Kristel; Hansmann, Florian; Elmarabet, Suliman Ahmed; Khan, Muhammad Akram; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner; Beineke, Andreas

    2015-11-01

    Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.

  11. A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

    Science.gov (United States)

    Mondanelli, Giada; Bianchi, Roberta; Pallotta, Maria Teresa; Orabona, Ciriana; Albini, Elisa; Iacono, Alberta; Belladonna, Maria Laura; Vacca, Carmine; Fallarino, Francesca; Macchiarulo, Antonio; Ugel, Stefano; Bronte, Vincenzo; Gevi, Federica; Zolla, Lello; Verhaar, Auke; Peppelenbosch, Maikel; Mazza, Emilia Maria Cristina; Bicciato, Silvio; Laouar, Yasmina; Santambrogio, Laura; Puccetti, Paolo; Volpi, Claudia; Grohmann, Ursula

    2017-02-21

    Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1(+) myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

  12. Myeloid derived suppressor cells (MDSCs are increased and exert immunosuppressive activity together with polymorphonuclear leukocytes (PMNs in chronic myeloid leukemia patients.

    Directory of Open Access Journals (Sweden)

    Cesarina Giallongo

    Full Text Available Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs, able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1 that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.

  13. Obesity induces a phenotypic switch in adipose tissue macrophage polarization.

    Science.gov (United States)

    Lumeng, Carey N; Bodzin, Jennifer L; Saltiel, Alan R

    2007-01-01

    Adipose tissue macrophages (ATMs) infiltrate adipose tissue during obesity and contribute to insulin resistance. We hypothesized that macrophages migrating to adipose tissue upon high-fat feeding may differ from those that reside there under normal diet conditions. To this end, we found a novel F4/80(+)CD11c(+) population of ATMs in adipose tissue of obese mice that was not seen in lean mice. ATMs from lean mice expressed many genes characteristic of M2 or "alternatively activated" macrophages, including Ym1, arginase 1, and Il10. Diet-induced obesity decreased expression of these genes in ATMs while increasing expression of genes such as those encoding TNF-alpha and iNOS that are characteristic of M1 or "classically activated" macrophages. Interestingly, ATMs from obese C-C motif chemokine receptor 2-KO (Ccr2-KO) mice express M2 markers at levels similar to those from lean mice. The antiinflammatory cytokine IL-10, which was overexpressed in ATMs from lean mice, protected adipocytes from TNF-alpha-induced insulin resistance. Thus, diet-induced obesity leads to a shift in the activation state of ATMs from an M2-polarized state in lean animals that may protect adipocytes from inflammation to an M1 proinflammatory state that contributes to insulin resistance.

  14. Caffeine plus nicotine improves motor function, spatial and non-spatial working memory and functional indices in BALB/c male mice.

    Science.gov (United States)

    Adeniyi, P A; Omatsuli, E P; Akinyemi, A J; Ishola, A O

    2016-12-01

    There is a greater prevalence of cigarette smoking among caffeine dependent individuals. This study therefore sought to assess the effect of nicotine and/or caffeine on some key biochemical indices and neurobehavioural parameters associated with brain function in male mice. Forty male BALB/c mice were divided into 4 groups of 10 animals each; Group A serve as the control and received normal saline (s.c), Group B received 2mg/kg body weight of nicotine (s.c), Group C received 2mg/kg body weight of caffeine (s.c) and Group D received 2mg/kg of nicotine and 2mg/kg of caffeine (s.c). The experiment lasted for 21 days, and then the animals were subjected to behavioral test. Thereafter the animals were sacrificed and their brain isolated for the determination of endothelial nitric oxide (NO) level, acetylcholinesterase (AChE), arginase (Arg) and adenosine deaminase (ADA) activities; as well as some antioxidant indices. Administration of nicotine or caffeine caused a significant (Pnicotine enhances caffeine cognitive properties through a significant increase in non-spatial working memory whereas; it was otherwise on the spatial working memory and motor coordination. Therefore, we can suggest from our present study that caffeine enhances the effect of nicotine either synergistically or additively on memory and motor function and some key biochemical indices associated with brain function in male mice.

  15. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  16. Evolution of urea transporters in vertebrates: adaptation to urea's multiple roles and metabolic sources.

    Science.gov (United States)

    LeMoine, Christophe M R; Walsh, Patrick J

    2015-06-01

    In the two decades since the first cloning of the mammalian kidney urea transporter (UT-A), UT genes have been identified in a plethora of organisms, ranging from single-celled bacteria to metazoans. In this review, focusing mainly on vertebrates, we first reiterate the multiple catabolic and anabolic pathways that produce urea, then we reconstruct the phylogenetic history of UTs, and finally we examine the tissue distribution of UTs in selected vertebrate species. Our analysis reveals that from an ancestral UT, three homologues evolved in piscine lineages (UT-A, UT-C and UT-D), followed by a subsequent reduction to a single UT-A in lobe-finned fish and amphibians. A later internal tandem duplication of UT-A occurred in the amniote lineage (UT-A1), followed by a second tandem duplication in mammals to give rise to UT-B. While the expected UT expression is evident in excretory and osmoregulatory tissues in ureotelic taxa, UTs are also expressed ubiquitously in non-ureotelic taxa, and in tissues without a complete ornithine-urea cycle (OUC). We posit that non-OUC production of urea from arginine by arginase, an important pathway to generate ornithine for synthesis of molecules such as polyamines for highly proliferative tissues (e.g. testis, embryos), and neurotransmitters such as glutamate for neural tissues, is an important evolutionary driving force for the expression of UTs in these taxa and tissues.

  17. Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Lifeng Xiong

    2016-03-01

    Full Text Available Antibacterial resistance to infectious diseases is a significant global concern for health care organizations; along with aging populations and increasing cancer rates, it represents a great burden for government healthcare systems. Therefore, the development of therapies against bacterial infection and cancer is an important strategy for healthcare research. Pathogenic bacteria and cancer have developed a broad range of sophisticated strategies to survive or propagate inside a host and cause infection or spread disease. Bacteria can employ their own metabolism pathways to obtain nutrients from the host cells in order to survive. Similarly, cancer cells can dysregulate normal human cell metabolic pathways so that they can grow and spread. One common feature of the adaption and disruption of metabolic pathways observed in bacterial and cancer cell growth is amino acid pathways; these have recently been targeted as a novel approach to manage bacterial infections and cancer therapy. In particular, arginine metabolism has been illustrated to be important not only for bacterial pathogenesis but also for cancer therapy. Therefore, greater insights into arginine metabolism of pathogenic bacteria and cancer cells would provide possible targets for controlling of bacterial infection and cancer treatment. This review will summarize the recent progress on the relationship of arginine metabolism with bacterial pathogenesis and cancer therapy, with a particular focus on arginase and arginine deiminase pathways of arginine catabolism.

  18. Mechanisms of Evodiamine Attenuating M2 Polarized Macrophages%吴茱萸碱抑制M2型巨噬细胞功能的机制研究

    Institute of Scientific and Technical Information of China (English)

    樊垒垒; 朱飞云; 崔琳; 王小晓; 张松江; 沈思; 朱明军; 王幼平

    2015-01-01

    目的:吴茱萸碱是中药吴茱萸重要生物碱成分之一,其具有明显的抗炎作用,但具体机制不清.研究显示瞬时受体电位香草醛亚型1受体(Transient Receptor Potential Vanilloid Type 1 Channel,TRPV1)对炎症反应具有明显的抑制作用,而吴茱萸碱可激活TRPV1受体.本研究以人单核细胞(THP-1)培养模型,探明吴茱萸碱对M2型巨噬细胞功能的影响及TRPV1受体参与该过程中的作用,从而阐明吴茱萸碱抑制炎症反应的分子生物机制.方法:在THP-1细胞培养模型上,观察吴茱萸碱对白介素-4(Interleukin-4,IL-4)诱导的M2型巨噬细胞功能的影响,以及特异性TRPV1受体拮抗剂Capsazepine(CAPZ)对该过程的影响,并分别利用ELISA、荧光定量PCR和western blot确定M2型巨噬细胞功能指标,其中包括TGF-β1的产生、Arginase-1和Mannose Receptor mRNA和蛋白的表达.结果:本研究发现吴茱萸碱明显抑制IL-4诱导的M2型巨噬细胞功能亚型,其主要表现为TGF-β1分泌产生下降(P<0.05),同时伴有Arginase-1和Mannose Receptor mRNA和蛋白表达的降低(P<0.05),以上结果可被特异性TRPV1受体拮抗剂CAPZ所阻断(P<0.05).结论:本研究显示吴茱萸碱通过激活TRPV1受体抑制M2型巨噬细胞功能反应,从而实现其抑制炎症反应的作用.%Evodiamine (EVO) was one of the important alkaloids extracted from Chinese herb Evodia rutaecarpa which possessed the ability of anti-inflammation.However,the mechanisms responsible for its anti-inflammation remained to be elucidated.Several studies had shown that the transient receptor potential vanilloid type 1 channel (TRPV1) had obvious inhibitory effects on inflammatory response; and EVO activated TRPV1.This study cultured THP-1 model for the exploration on effect of EVO on M2 polarized macrophages as well as the role of TRPV1,in order to illustrate the molecular biological mechanisms of anti-inflammation effect by EVO.Based on the THP-1 cell culture model

  19. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

    Science.gov (United States)

    Wu, Dianhui; Li, Xiaomin; Lu, Jian; Chen, Jian; Zhang, Liang; Xie, Guangfa

    2016-01-01

    Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine.

  20. In vivo immunomodulatory effect of the lectin from edible mushroom Agaricus bisporus.

    Science.gov (United States)

    Ditamo, Yanina; Rupil, Lucia L; Sendra, Victor G; Nores, Gustavo A; Roth, German A; Irazoqui, Fernando J

    2016-01-01

    Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.

  1. Dendrosomal curcumin suppresses metastatic breast cancer in mice by changing m1/m2 macrophage balance in the tumor microenvironment.

    Science.gov (United States)

    Shiri, Sadaf; Alizadeh, Ali Mohammad; Baradaran, Behzad; Farhanghi, Baharak; Shanehbandi, Dariush; Khodayari, Saeed; Khodayari, Hamid; Tavassoli, Abbas

    2015-01-01

    Curcumin, a lipid-soluble compound extracted from the plant Curcuma Longa, has been found to exert immunomodulatory effects via macrophages. However, most studies focus on the low bioavailability issue of curcumin by nano and microparticles, and thus the role of macrophages in the anticancer mechanism of curcumin has received little attention so far. We have previously shown the potential biocompatibility, biodegradability and anti-cancer effects of dendrosomal curcumin (DNC). In this study, twenty-seven BALB/c mice were equally divided into control as well as 40 and 80 mg/kg groups of DNC to investigate the involvement of macrophages in the antitumor effects of curcumin in a typical animal model of metastatic breast cancer. At the end of intervention, the tumor volume and weight were significantly reduced in DNC groups compared to control (PDNC increased the expression of STAT4 and IL-12 genes in tumor and spleen tissues in comparison with control (PDNC decreased STAT3, IL-10 and arginase I gene expression (P<0.05), indicating low levels of M2 macrophage. The results confirm the role of macrophages in the protective effects of dendrosomal curcumin against metastatic breast cancer in mice.

  2. [The role of nitric oxide and superoxide synthesis in protective mechanism of ecdysterone in the heart mitochondria of rats with streptozotocin-induced diabetes].

    Science.gov (United States)

    Korkach, Iu P; Rudyk, O V; Kotsiuruba, A V; Prysiazhna, O D; Sahach, V F

    2007-01-01

    This study evaluated generation of O2*- and NO in heart mitochondria isolated from 9-week old streptozotocin (STZ)-induced diabetic rats and the effect of ecdysterone treatment on these parameters. Mitochondria isolated from 9-week old placebo-treated rats were used as control. Several parameters were evaluated: O2*- production, the levels of stable NO metabolites nitrate, nitrite and total nitrosothiols, the level of bilirubine (as marker of CO generation), inducible (iNOS) and constitutive (nNOS) mtNOS, NADH- dependent nitrate reductase (NR) and inducible arginase II (AII) activity. We observed that diabetes was accompanied by a significant decrease in nNOS activity, nitrite, total nitrosothiols and bilirubine content while iNOS, NR and AII activity, as well as O2*- generation was increased in heart mitochondria. Ecdysterone treatment normalized the levels of stable NO metabolites, ability to generate superoxide, iNOS and nNOS activity, but not bilirubine level, NR and AII activity. These results suggest that ecdysterone treatment attenuates diabetes-induced mitochondrial alterations protecting against oxidative and nitrosative stresses. Thus, ecdysterone therapy, besides its well known importance in the maintenance of glycemic control, may help to protect against mitochondrial dysfunction associated to several age-related disorders.

  3. Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration.

    Science.gov (United States)

    Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

    2015-04-24

    Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors.

  4. Carbon availability triggers fungal nitrogen uptake and transport in arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Fellbaum, Carl R; Gachomo, Emma W; Beesetty, Yugandhar; Choudhari, Sulbha; Strahan, Gary D; Pfeffer, Philip E; Kiers, E Toby; Bücking, Heike

    2012-02-14

    The arbuscular mycorrhizal (AM) symbiosis, formed between the majority of land plants and ubiquitous soil fungi of the phylum Glomeromycota, is responsible for massive nutrient transfer and global carbon sequestration. AM fungi take up nutrients from the soil and exchange them against photosynthetically fixed carbon (C) from the host. Recent studies have demonstrated that reciprocal reward strategies by plant and fungal partners guarantee a "fair trade" of phosphorus against C between partners [Kiers ET, et al. (2011) Science 333:880-882], but whether a similar reward mechanism also controls nitrogen (N) flux in the AM symbiosis is not known. Using mycorrhizal root organ cultures, we manipulated the C supply to the host and fungus and followed the uptake and transport of N sources in the AM symbiosis, the enzymatic activities of arginase and urease, and fungal gene expression in the extraradical and intraradical mycelium. We found that the C supply of the host plant triggers the uptake and transport of N in the symbiosis, and that the increase in N transport is orchestrated by changes in fungal gene expression. N transport in the symbiosis is stimulated only when the C is delivered by the host across the mycorrhizal interface, not when C is supplied directly to the fungal extraradical mycelium in the form of acetate. These findings support the importance of C flux from the root to the fungus as a key trigger for N uptake and transport and provide insight into the N transport regulation in the AM symbiosis.

  5. Oral administration of an enzymatic protein hydrolysate from the green microalga Chlorella vulgaris enhances the nutritional recovery of malnourished mice.

    Science.gov (United States)

    Morris, Humberto J; Carrillo, Olimpia V; Alonso, María E; Bermúdez, Rosa C; Almarales, Ángel; Llauradó, Gabriel; Lebeque, Yamila; Fontaine, Roberto

    2011-12-01

    This study examined the effects of oral administration of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris (Cv-PH) on the nutritional recovery of malnourished Balb/c mice after a 3-day fasting period. Mice were refed with commercial diet supplemented or not supplemented with Cv-PH (500 mg/kg) for 8 days. Regardless of the diet used during refeeding, animal body weights and serum protein concentrations did not differ between groups. Mice given Cv-PH had a significant increase in hemoglobin concentrations. Most serum amino acid levels were similar in the control and Cv-PH animals. Starved mice refed with Cv-PH showed normal liver functions, as judged by liver weight, protein concentration, and the enzymatic activities of cholinesterase and arginase. Cv-PH increased DNA, protein content, and gut-mucosal weight. In addition, brush-border oligosaccharidase activities were also higher in the Cv-PH group. These findings suggest that Chlorella protein hydrolysate can be used to develop specific formulations suitable for pharmacologic nutrition.

  6. Looking downstream: the role of cyclic AMP-regulated genes in axonal regeneration

    Directory of Open Access Journals (Sweden)

    Mustafa M Siddiq

    2015-06-01

    Full Text Available Elevation of intracellular cyclic AMP (cAMP levels has proven to be one of the most effective means of overcoming inhibition of axonal regeneration by myelin-associated inhibitors such as myelin-associated glycoprotein, Nogo, and oligodendrocyte myelin glycoprotein. Pharmacological manipulation of cAMP through the administration of dibutyryl cAMP or rolipram leads to enhanced axonal growth both in vivo and in vitro, and importantly, upregulation of cAMP within dorsal root ganglion neurons is responsible for the conditioning lesion effect, which indicates that cAMP plays a significant role in the endogenous mechanisms that promote axonal regeneration. The effects of cAMP are transcription-dependent and are mediated through the activation of protein kinase A and the transcription factor CREB. This leads to the induction of a variety of genes, several of which have been shown to overcome myelin-mediated inhibition in their own right. In this review, we will highlight the pro-regenerative effects of arginase I, interleukin-6, secretory leukocyte protease inhibitor, and metallothionein-I/II, and discuss their potential for therapeutic use in spinal cord injury.

  7. PET Imaging with [(18)F]FSPG Evidences the Role of System xc(-) on Brain Inflammation Following Cerebral Ischemia in Rats.

    Science.gov (United States)

    Domercq, Maria; Szczupak, Boguslaw; Gejo, Jon; Gómez-Vallejo, Vanessa; Padro, Daniel; Gona, Kiran Babu; Dollé, Frédéric; Higuchi, Makoto; Matute, Carlos; Llop, Jordi; Martín, Abraham

    2016-01-01

    In vivo Positron Emission Tomography (PET) imaging of the cystine-glutamate antiporter (system xc(-)) activity with [(18)F]FSPG is meant to be an attractive tool for the diagnosis and therapy evaluation of brain diseases. However, the role of system xc(-) in cerebral ischemia and its involvement in inflammatory reaction has been scarcely explored. In this work, we report the longitudinal investigation of the neuroinflammatory process following transient middle cerebral artery occlusion (MCAO) in rats using PET with [(18)F]FSPG and the translocator protein (TSPO) ligand [(18)F]DPA-714. In the ischemic territory, [(18)F]FSPG showed a progressive binding increase that peaked at days 3 to 7 and was followed by a progressive decrease from days 14 to 28 after reperfusion. In contrast, [(18)F]DPA-714 evidenced maximum binding uptake values over day 7 after reperfusion. Ex vivo immnunohistochemistry confirmed the up-regulation of system xc(-) in microglial cells and marginally in astrocytes. Inhibition of system xc(-) with sulfasalazine and S-4-CPG resulted in increased arginase (anti-inflammatory M2 marker) expression at day 7 after ischemia, together with a decrease in TSPO and microglial M1 proinflammatory markers (CCL2, TNF and iNOS) expression. Taken together, these results suggest that system xc(-) plays a key role in the inflammatory reaction underlying experimental stroke.

  8. Ozone-induced injury and oxidative stress in bronchiolar epithelium are associated with altered pulmonary mechanics.

    Science.gov (United States)

    Sunil, Vasanthi R; Vayas, Kinal N; Massa, Christopher B; Gow, Andrew J; Laskin, Jeffrey D; Laskin, Debra L

    2013-06-01

    In these studies, we analyzed the effects of ozone on bronchiolar epithelium. Exposure of rats to ozone (2 ppm, 3 h) resulted in rapid (within 3 h) and persistent (up to 72 h) histological changes in the bronchiolar epithelium, including hypercellularity, loss of cilia, and necrotizing bronchiolitis. Perivascular edema and vascular congestion were also evident, along with a decrease in Clara cell secretory protein in bronchoalveolar lavage, which was maximal 24 h post-exposure. Ozone also induced the appearance of 8-hydroxy-2'-deoxyguanosine, Ym1, and heme oxygenase-1 in the bronchiolar epithelium. This was associated with increased expression of cleaved caspase-9 and beclin-1, indicating initiation of apoptosis and autophagy. A rapid and persistent increase in galectin-3, a regulator of epithelial cell apoptosis, was also observed. Following ozone exposure (3-24 h), increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and arginase-1 was noted in bronchiolar epithelium. Ozone-induced injury and oxidative stress in bronchiolar epithelium were linked to methacholine-induced alterations in pulmonary mechanics. Thus, significant increases in lung resistance and elastance, along with decreases in lung compliance and end tidal volume, were observed at higher doses of methacholine. This indicates that ozone causes an increase in effective stiffness of the lung as a consequence of changes in the conducting airways. Collectively, these studies demonstrate that bronchiolar epithelium is highly susceptible to injury and oxidative stress induced by acute exposure to ozone; moreover, this is accompanied by altered lung functioning.

  9. Pretreatment optimization of the biomass of Microcystis aeruginosa for efficient bioethanol production.

    Science.gov (United States)

    Khan, Muhammad Imran; Lee, Moon Geon; Shin, Jin Hyuk; Kim, Jong Deog

    2017-12-01

    Microalgae are considered to be the future promising sources of biofuels and bio products. The algal carbohydrates can be fermented to bioethanol after pretreatment process. Efficient pretreatment of the biomass is one of the major requirements for commercialization of the algal based biofuels. In present study the microalga, M. aeruginsa was used for pretreatment optimization and bioethanol production. Treatment of algal biomass with CaO before acid and/or enzymatic hydrolysis enhanced the degradation of algal cells. Monomeric sugars yield was increased more than twice when biomass was pretreated with CaO. Similarly, an increase was noted in the amount of fermentable sugars when biomass was subjected to invertase saccharification after acid or lysozyme pretreatment. Highest yield of fermentable sugars (16 mM/ml) in the centrifuged algal juice was obtained. 4 Different microorganisms' species were used individually and in combination for converting centrifuged algal juice to bioethanol. Comparatively higher yield of bioethanol (60 mM/ml) was obtained when the fermenter microorganisms were used in combination. The results demonstrated that M. arginase biomass can be efficiently pretreated to get higher yield of fermentable sugars for enhanced yield of bioethanol production.

  10. Nitrite to nitrate molar ratio is inversely proportional to oxidative cell damages and granulocytic apoptosis at the wound site following cutaneous injury in rats.

    Science.gov (United States)

    Zunić, Gordana; Colić, Miodrag; Vuceljić, Marina

    2009-06-01

    Nitric oxide (NO) metabolism in response to the inflammatory cell infiltration and their apoptosis at the wound site, using a model of subcutaneously implanted sponges in Albino Oxford rats, were examined. The injured animals were sacrificed at days 1, 2 and 3 after the injury. Nitrites, nitrates (final products of NO metabolism), malondialdehyde (an indicator of oxidative cell damages), urea (product of arginase activity) and other parameters were measured both in plasma and wound fluid samples. Nitrite to nitrate molar ratio and sum of nitrites and nitrates (NO(x)) were calculated. The total cell numbers were at similar level throughout the examined period, but a gradual decrease of viable granulocytes, mainly due to the increased apoptosis, and the increase of monocyte-macrophage number occurred after the second day. A gradual increase of wound fluid nitrates, NO(x) and malondialdehyde suggested the increases of both NO and free oxygen radicals production. Interestingly, wound fluid nitrites peaked at the first day decreasing to the corresponding plasma levels thereafter. Wound fluid nitrite to nitrate molar ratio gradually decreased and negatively correlated both with the number of apoptotic cells (r= -0.752, poxidative cell damages and cell apoptosis at the wound site early after the cutaneous wound. Moreover, the obtained findings suggest that measurement of both nitrites and nitrates contribute to better insight into overall wound NO metabolism.

  11. Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism

    Science.gov (United States)

    Weinstein, L. H.; Osmeloski, J. F.; Wettlaufer, S. H.; Galston, A. W.

    1987-01-01

    In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.

  12. Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension.

    Science.gov (United States)

    Karmouty-Quintana, Harry; Philip, Kemly; Acero, Luis F; Chen, Ning-Yuan; Weng, Tingting; Molina, Jose G; Luo, Fayong; Davies, Jonathan; Le, Ngoc-Bao; Bunge, Isabelle; Volcik, Kelly A; Le, Thanh-Thuy T; Johnston, Richard A; Xia, Yang; Eltzschig, Holger K; Blackburn, Michael R

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2B(f/f)-LysM(Cre)) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2B(f/f)-LysM(Cre) mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.

  13. Monocyte Differentiation towards Protumor Activity Does Not Correlate with M1 or M2 Phenotypes

    Science.gov (United States)

    Chimal-Ramírez, G. Karina; Espinoza-Sánchez, Nancy Adriana; Chávez-Sánchez, Luis; Arriaga-Pizano, Lourdes

    2016-01-01

    Macrophages facilitate breast cancer progression. Macrophages were initially classified as M1 or M2 based on their distinct metabolic programs and then expanded to include antitumoral (M1) and protumoral (M2) activities. However, it is still uncertain what markers define the pro- and antitumoral phenotypes and what conditions lead to their formation. In this study, monocytic cell lines and primary monocytes were subjected to commonly reported protocols of M1/M2 polarization and conditions known to engage monocytes into protumoral functions. The results showed that only IDO enzyme and CD86 M1 markers were upregulated correlating with M1 polarization. TNF-α, CCR7, IL-10, arginase I, CD36, and CD163 were expressed indistinguishably from M1 or M2 polarization. Similarly, protumoral engaging resulted in upregulation of both M1 and M2 markers, with conditioned media from the most aggressive breast cancer cell line promoting the greatest changes. In spite of the mixed phenotype, M1-polarized macrophages exhibited the highest expression/secretion of inflammatory mediators, many of which have previously been associated with breast cancer aggressiveness. These data argue that although the existence of protumoral macrophages is unquestionable, their associated phenotypes and the precise conditions driving their formation are still unclear, and those conditions may need both M1 and M2 stimuli. PMID:27376091

  14. The macrophage galactose-type lectin-1 (MGL1) recognizes Taenia crassiceps antigens, triggers intracellular signaling, and is critical for resistance to this infection.

    Science.gov (United States)

    Montero-Barrera, Daniel; Valderrama-Carvajal, Héctor; Terrazas, César A; Rojas-Hernández, Saúl; Ledesma-Soto, Yadira; Vera-Arias, Laura; Carrasco-Yépez, Maricela; Gómez-García, Lorena; Martínez-Saucedo, Diana; Becerra-Díaz, Mireya; Terrazas, Luis I

    2015-01-01

    C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1(-/-) mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1(-/-) macrophages. Following T. crassiceps infection, MGL1(-/-) mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1(-/-) mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1(-/-) macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance.

  15. Pair housing reverses post-stroke depressive behavior in mice.

    Science.gov (United States)

    Verma, Rajkumar; Friedler, Brett D; Harris, Nia M; McCullough, Louise D

    2014-08-01

    Social isolation (SI) has been linked epidemiologically to high rates of morbidity and mortality following stroke. In contrast, strong social support enhances recovery and lowers stroke recurrence. However, the mechanism by which social support influences stroke recovery has not been adequately explored. The goal of this study was to examine the effect of post-stroke pair housing and SI on behavioral phenotypes and chronic functional recovery in mice. Young male mice were paired for 14 days before a 60 min transient middle cerebral artery occlusion (MCAO) or sham surgery and assigned to various housing environments immediately after stroke. Post-stroke mice paired with either a sham or stroke partner showed significantly higher (Preduced immobility in the tail suspension test compared to isolated cohorts. Pair-housed stroked mice demonstrated significantly reduced cerebral atrophy after 6 weeks (17.5 ± 1.5% in PH versus 40.8 ± 1.3% in SI; Preduces avolitional and anhedonic behavior. Pair housing reduced serum IL-6 and enhanced peri-infarct microglia arginase-1 expression. Social interaction reduces post-stroke depression and improves functional recovery.

  16. Acquired resistance to innate immune clearance promotes Klebsiella pneumoniae ST258 pulmonary infection

    Science.gov (United States)

    Ahn, Danielle; Peñaloza, Hernán; Wang, Zheng; Wickersham, Matthew; Parker, Dane; Patel, Purvi; Koller, Antonius; Chen, Emily I.; Bueno, Susan M.; Uhlemann, Anne-Catrin; Prince, Alice

    2016-01-01

    Adaptive changes in the genome of a locally predominant clinical isolate of the multidrug-resistant Klebsiella pneumoniae ST258 (KP35) were identified and help to explain the selection of this strain as a successful pulmonary pathogen. The acquisition of 4 new ortholog groups, including an arginine transporter, enabled KP35 to outcompete related ST258 strains lacking these genes. KP35 infection elicited a monocytic response, dominated by Ly6Chi monocytic myeloid-derived suppressor cells that lacked phagocytic capabilities, expressed IL-10, arginase, and antiinflammatory surface markers. In comparison with other K. pneumoniae strains, KP35 induced global changes in the phagocytic response identified with proteomics, including evasion of Ca2+ and calpain activation necessary for phagocytic killing, confirmed in functional studies with neutrophils. This comprehensive analysis of an ST258 K. pneumoniae isolate reveals ongoing genetic adaptation to host microenvironments and innate immune clearance mechanisms that complements its repertoire of antimicrobial resistance genes and facilitates persistence in the lung. PMID:27777978

  17. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway.

    Science.gov (United States)

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-07

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by N(ω)-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension.

  18. The Occurrence and Toxicity of Indospicine to Grazing Animals

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    Mary T. Fletcher

    2015-07-01

    Full Text Available Indospicine is a non-proteinogenic amino acid which occurs in Indigofera species with widespread prevalence in grazing pastures across tropical Africa, Asia, Australia, and the Americas. It accumulates in the tissues of grazing livestock after ingestion of Indigofera. It is a competitive inhibitor of arginase and causes both liver degeneration and abortion. Indospicine hepatoxicity occurs universally across animal species but the degree varies considerably between species, with dogs being particularly sensitive. The magnitude of canine sensitivity is such that ingestion of naturally indospicine-contaminated horse and camel meat has caused secondary poisoning of dogs, raising significant industry concern. Indospicine impacts on the health and production of grazing animals per se has been less widely documented. Livestock grazing Indigofera have a chronic and cumulative exposure to this toxin, with such exposure experimentally shown to induce both hepatotoxicity and embryo-lethal effects in cattle and sheep. In extensive pasture systems, where animals are not closely monitored, the resultant toxicosis may well occur after prolonged exposure but either be undetected, or even if detected not be attributable to a particular cause. Indospicine should be considered as a possible cause of animal poor performance, particularly reduced weight gain or reproductive losses, in pastures where Indigofera are prevalent.

  19. Fungal lectin of Peltigera canina induces chemotropism of compatible Nostoc cells by constriction-relaxation pulses of cyanobiont cytoskeleton.

    Science.gov (United States)

    Díaz, Eva Maria; Vicente-Manzanares, Miguel; Sacristan, Mara; Vicente, Carlos; Legaz, Maria-Estrella

    2011-10-01

    A glycosylated arginase acting as a fungal lectin from Peltigera canina is able to produce recruitment of cyanobiont Nostoc cells and their adhesion to the hyphal surface. This implies that the cyanobiont would develop organelles to motility towards the chemoattractant. However when visualized by transmission electron microscopy, Nostoc cells recently isolated from P. canina thallus do not reveal any motile, superficial organelles, although their surface was covered by small spindles and serrated layer related to gliding. The use of S-(3,4-dichlorobenzyl)isothiourea, blebbistatin, phalloidin and latrunculin A provide circumstantial evidence that actin microfilaments rather than MreB, the actin-like protein from prokaryota, and, probably, an ATPase which develops contractile function similar to that of myosin II, are involved in cell motility. These experimental facts, the absence of superficial elements (fimbriae, pili or flagellum) related to cell movement, and the appearance of sunken cells during of after movement verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin act as a chemoattractant.

  20. A Lichen Lectin Specifically Binds to the α-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae

    Science.gov (United States)

    Sacristán, Mara; Millanes, Ana-María; Legaz, María-Estrella

    2006-01-01

    A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with α-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of β-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner. PMID:19521472

  1. A Lichen Lectin Specifically Binds to the alpha-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae.

    Science.gov (United States)

    Sacristán, Mara; Millanes, Ana-María; Legaz, María-Estrella; Vicente, Carlos

    2006-01-01

    A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with alpha-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of beta-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.

  2. Cell-extrinsic effects of tumor ER stress imprint myeloid dendritic cells and impair CD8⁺ T cell priming.

    Science.gov (United States)

    Mahadevan, Navin R; Anufreichik, Veronika; Rodvold, Jeffrey J; Chiu, Kevin T; Sepulveda, Homero; Zanetti, Maurizio

    2012-01-01

    Tumor-infiltrating myeloid cells, such as dendritic cells (BMDC), are key regulators of tumor growth. However, the tumor-derived signals polarizing BMDC to a phenotype that subverts cell-mediated anti-tumor immunity have yet to be fully elucidated. Addressing this unresolved problem we show that the tumor unfolded protein response (UPR) can function in a cell-extrinsic manner via the transmission of ER stress (TERS) to BMDC. TERS-imprinted BMDC upregulate the production of pro-inflammatory, tumorigenic cytokines but also the immunosuppressive enzyme arginase. Importantly, they downregulate cross-presentation of high-affinity antigen and fail to effectively cross-prime CD8(+) T cells, causing T cell activation without proliferation and similarly dominantly suppress cross-priming by bystander BMDC. Lastly, TERS-imprinted BMDC facilitate tumor growth in vivo with fewer tumor-infiltrating CD8(+) T cells. In sum, we demonstrate that tumor-borne ER stress imprints ab initio BMDC to a phenotype that recapitulates several of the inflammatory/suppressive characteristics ascribed to tumor-infiltrating myeloid cells, highlighting the tumor UPR as a critical controller of anti-tumor immunity and a new target for immune modulation in cancer.

  3. Cell-extrinsic effects of tumor ER stress imprint myeloid dendritic cells and impair CD8⁺ T cell priming.

    Directory of Open Access Journals (Sweden)

    Navin R Mahadevan

    Full Text Available Tumor-infiltrating myeloid cells, such as dendritic cells (BMDC, are key regulators of tumor growth. However, the tumor-derived signals polarizing BMDC to a phenotype that subverts cell-mediated anti-tumor immunity have yet to be fully elucidated. Addressing this unresolved problem we show that the tumor unfolded protein response (UPR can function in a cell-extrinsic manner via the transmission of ER stress (TERS to BMDC. TERS-imprinted BMDC upregulate the production of pro-inflammatory, tumorigenic cytokines but also the immunosuppressive enzyme arginase. Importantly, they downregulate cross-presentation of high-affinity antigen and fail to effectively cross-prime CD8(+ T cells, causing T cell activation without proliferation and similarly dominantly suppress cross-priming by bystander BMDC. Lastly, TERS-imprinted BMDC facilitate tumor growth in vivo with fewer tumor-infiltrating CD8(+ T cells. In sum, we demonstrate that tumor-borne ER stress imprints ab initio BMDC to a phenotype that recapitulates several of the inflammatory/suppressive characteristics ascribed to tumor-infiltrating myeloid cells, highlighting the tumor UPR as a critical controller of anti-tumor immunity and a new target for immune modulation in cancer.

  4. Histological and biochemical effects of cigarette smoke on lungs.

    Science.gov (United States)

    Ozan, E; Kükner, A; Canpolat, L; Oner, H; Gezen, M R; Yilmaz, S; Ozan, S

    2001-01-01

    In this study, rats were made to inhale cigarette smoke in a specifically prepared container for different periods. The lung tissue samples of the subjects were examined by light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Malonaldehyde, one of the free oxygen radicals was determined in lungs and plasma. The catalase activity level of erythrocyte and arginase levels were determined. Three groups were formed. The rats in the Ist and IInd groups were made to inhale cigarette smoke for 30 and 60 minutes a day for a total period of 3 months. Control group, the rats in the IIIrd group (controls) were made to inhale clean air during the same periods. An increase in the number of macrophages was observed in the pulmonary tissue of the exposed groups. Especially in the group that inhaled the smoke for long periods, the number of macrophages and the inclusion bodies contained in them increased. These differences could easily be observed in TEM studies. In the light microscopy and SEM observations, it arouse attention that the alveolar macrophages occurred as sets and their activation increased. Depending on the length of the exposure to cigarette smoke, an increase in the number of macrophages was observed. Statistically significant increases were determined in the malonaldehyde levels of pulmonary tissue and plasma when compared to the control group. Besides significant increases were found in the catalase activity levels of erythrocytes in the experimental groups.

  5. IL-4Rα-dependent alternative activation of macrophages is not decisive for Mycobacterium tuberculosis pathology and bacterial burden in mice.

    Directory of Open Access Journals (Sweden)

    Reto Guler

    Full Text Available Classical activation of macrophages (caMph or M1 is crucial for host protection against Mycobacterium tuberculosis (Mtb infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2 are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB, we infected mice deficient for IL-4 receptor α on macrophages (LysMcreIL-4Rα-/lox with Mtb. We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT and LysMcreIL-4Rα-/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS and Arginase 1 (Arg1, well-established markers for M1/M2 macrophages among the Mtb-infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.

  6. Hematological disorders and pulmonary hypertension

    Science.gov (United States)

    Mathew, Rajamma; Huang, Jing; Wu, Joseph M; Fallon, John T; Gewitz, Michael H

    2016-01-01

    Pulmonary hypertension (PH), a serious disorder with a high morbidity and mortality rate, is known to occur in a number of unrelated systemic diseases. Several hematological disorders such as sickle cell disease, thalassemia and myeloproliferative diseases develop PH which worsens the prognosis. Associated oxidant injury and vascular inflammation cause endothelial damage and dysfunction. Pulmonary vascular endothelial damage/dysfunction is an early event in PH resulting in the loss of vascular reactivity, activation of proliferative and antiapoptotic pathways leading to vascular remodeling, elevated pulmonary artery pressure, right ventricular hypertrophy and premature death. Hemolysis observed in hematological disorders leads to free hemoglobin which rapidly scavenges nitric oxide (NO), limiting its bioavailability, and leading to endothelial dysfunction. In addition, hemolysis releases arginase into the circulation which converts L-arginine to ornithine, thus bypassing NO production. Furthermore, treatments for hematological disorders such as immunosuppressive therapy, splenectomy, bone marrow transplantation, and radiation have been shown to contribute to the development of PH. Recent studies have shown deregulated iron homeostasis in patients with cardiopulmonary diseases including pulmonary arterial hypertension (PAH). Several studies have reported low iron levels in patients with idiopathic PAH, and iron deficiency is an important risk factor. This article reviews PH associated with hematological disorders and its mechanism; and iron homeostasis and its relevance to PH. PMID:28070238

  7. Structure-Property Analysis of L-Ornithine and Its Substituted Analogues%L-鸟氨酸及其取代衍生物的结构和性质分析

    Institute of Scientific and Technical Information of China (English)

    MOHAMED IMRAN P.K.; SUBRAMANI K.

    2009-01-01

    L-ornithine is a metabolic product of L-arginine and is found in the liver and other tissues after the enzyme arginase in the urea cycle catalyses the reaction. The presence of L-ornithine has an influence on certain biochemical activities such as the proliferation of collagen production and also has an effect on airway responsiveness. Therefore, it has a role to play in the availability of nitric oxide (NO). A brief reactivity study was carried out for various substituents at the α-C atom of this amino acid. Substituents such as chlorine and fluorine affect the reactivity of the entire molecule and may alter the properties of the amino acid. To understand the properties of molecule, a detailed study was done at the density functional theory (DFT) level and compared with ab initio calculations. Fukui functions were invoked to assess reactivity and stability. The descriptive properties were correlated to logP or pK_a Using available software to help build a model to quantitatively assess properties and reactivity. Results showed a random reactivity pattern upon halogen substitution and this indicates the need to exercise caution during docking and reactivity studies.

  8. High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

    Directory of Open Access Journals (Sweden)

    Koop Ben F

    2010-05-01

    Full Text Available Abstract Background Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma indices. Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR. qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response. Results Granulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-β (p = 0.001, IL-17A (p = 0.007 and its receptor (IL-17AR (p = 0.009 representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of TH1 T cell lineage (IFN-γ, CD4 or TH2 (GATA-3

  9. Protective role of G-CSF in dextran sulfate sodium-induced acute colitis through generating gut-homing macrophages.

    Science.gov (United States)

    Meshkibaf, Shahab; Martins, Andrew J; Henry, Garth T; Kim, Sung Ouk

    2016-02-01

    Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine best known for its role in promoting the generation and function of neutrophils. G-CSF is also found to be involved in macrophage generation and immune regulation; however, its in vivo role in immune homeostasis is largely unknown. Here, we examined the role of G-CSF in dextran sulfate sodium (DSS)-induced acute colitis using G-CSF receptor-deficient (G-CSFR(-/-)) mice. Mice were administered with 1.5% DSS in drinking water for 5days, and the severity of colitis was measured for the next 5days. GCSFR(-/-) mice were more susceptible to DSS-induced colitis than G-CSFR(+/+) or G-CSFR(-/+) mice. G-CSFR(-/-) mice harbored less F4/80(+) macrophages, but a similar number of neutrophils, in the intestine. In vitro, bone marrow-derived macrophages prepared in the presence of both G-CSF and macrophage colony-stimulating factor (M-CSF) (G-BMDM) expressed higher levels of regulatory macrophage markers such as programmed death ligand 2 (PDL2), CD71 and CD206, but not in arginase I, transforming growth factor (TGF)-β, Ym1 (chitinase-like 3) and FIZZ1 (found in inflammatory zone 1), and lower levels of inducible nitric oxide synthase (iNOS), CD80 and CD86 than bone marrow-derived macrophages prepared in the presence of M-CSF alone (BMDM), in response to interleukin (IL)-4/IL-13 and lipopolysaccharide (LPS)/interferon (IFN)-γ, respectively. Adoptive transfer of G-BMDM, but not BMDM, protected G-CSFR(-/-) mice from DSS-induced colitis, and suppressed expression of tumor necrosis factor (TNF)-α, IL-1β and iNOS in the intestine. These results suggest that G-CSF plays an important role in preventing colitis, likely through populating immune regulatory macrophages in the intestine.

  10. Preferential magnetic nanoparticle uptake by bone marrow derived macrophages sub-populations: effect of surface coating on polarization, toxicity, and in vivo MRI detection

    Energy Technology Data Exchange (ETDEWEB)

    Al Faraj, Achraf, E-mail: aalfaraj@ksu.edu.sa [College of Applied Medical Sciences, King Saud University, Molecular and Cellular Imaging Lab, Department of Radiological Sciences (Saudi Arabia)

    2013-07-15

    Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of different diseases, which make them attractive vehicles to deliver contrast agents or drugs for diagnostic or therapeutic purposes. In this study, the effect of polyethylene glycol functionalization of magnetic iron oxide nanoparticles and their further surface modification with carboxylic groups on bone marrow-derived M1 and M2 macrophages phenotype, labeling efficiency, uptake mechanism, biocompatibility, and their in vivo MR detection was assessed. An enhanced labeling efficiency was observed for carboxylic surface-modified superparamagnetic iron oxide (SPIO) compared to PEGylated SPIO and to a higher extent to plain SPIO along with a higher uptake by M2 subsets. Magnetic nanoparticles were found located in the periphery of the vesicles dispersed in the cytoplasm in TEM. Investigation of the labeling mechanism by inhibiting different uptake pathways revealed that endocytosis via scavenger receptor A, a process known to be clathrin mediated, plays a central role in the cellular uptake kinetics of both macrophages subpopulations. Biocompatibility evaluation showed no variation in cell viability and mitochondrial membrane potential with a low release of ROS. Flow cytometry and measurement of iNOS and Arginase 1 activity as marker of M1 and M2 macrophages polarization confirmed that magnetic labeling of macrophages subsets did not affect their polarization. In addition, no variation was observed in the biodistribution of magnetic iron oxide-labeled M1 and M2 macrophages subsets when monitored using noninvasive magnetic resonance imaging with a better detection for the enhanced SPIO-PEG-COOH-labeled cells.

  11. Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Tian; Wang, Chenlong; Chen, Xuewei; Duan, Chenfan; Zhang, Xiaoyan [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Tang, Tian [Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Chen, Honglei [Department of Pathology and Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yue, Jiang [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Li, Ying, E-mail: lyying0@163.com [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

    2015-07-15

    Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50 mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing the coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy. - Highlights: • Dopamine induces tumor growth inhibition and vascular normalization in rat C6 glioma. • Dopamine switches macrophage phenotype from M2 to M1. • Dopamine-induced vascular normalization is mediated by macrophage polarization. • Dopamine is a promising agent targeting the microvasculature in tumor

  12. Vegetable oil induced inflammatory response by altering TLR-NF-κB signalling, macrophages infiltration and polarization in adipose tissue of large yellow croaker (Larimichthys crocea).

    Science.gov (United States)

    Tan, Peng; Dong, Xiaojing; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-12-01

    High level of vegetable oil (VO) in diets could induce strong inflammatory response, and thus decrease nonspecific immunity and disease resistance in most marine fish species. The present study was conducted to investigate whether dietary VO could exert these anti-immunological effects by altering TLR-NF-κB signalling, macrophages infiltration and polarization in adipose tissue of large yellow croaker (Larimichthys crocea). Three iso-nitrogenous and iso-lipid diets with 0% (FO, fish oil, the control), 50% (FV, fish oil and vegetable oil mixed) and 100% (VO, vegetable oil) vegetable oil were fed to fish with three replicates for ten weeks. The results showed that activities of respiratory burst (RB) and alternative complement pathway (ACP), as well as disease resistance after immune challenge were significantly decreased in large yellow croaker fed VO diets compared to FO diets. Inflammatory response of experimental fish was markedly elevated by VO reflected by increase of pro-inflammatory cytokines (IL1β and TNFα) and decrease of anti-inflammatory cytokine (arginase I and IL10) genes expression. TLR-related genes expression, nucleus p65 protein, IKKα/β and IκBα phosphorylation were all significantly increased in the AT of large yellow croaker fed VO diets. Moreover, the expression of macrophage infiltration marker proteins (cluster of differentiation 68 [CD68] and colony-stimulating factor 1 receptor [CSF1R]) was significantly increased while the expression of anti-inflammatory M2 macrophage polarization marker proteins (macrophage mannose receptor 1 [MRC1] and cluster of differentiation 209 [CD209]) was significantly decreased in the AT of large yellow croaker fed VO diets. In conclusion, VO could induce inflammatory responses by activating TLR-NF-κB signalling, increasing macrophage infiltration into adipose tissue and polarization of macrophage in large yellow croaker.

  13. HIPERAMONEMIA NEONATAL CAUSADA POR DEFECTOS DEL CICLO DE LA UREA Neonatal hyperammonemia in urea cycle disorders patients

    Directory of Open Access Journals (Sweden)

    Yolanda Cifuentes C

    2010-12-01

    Full Text Available Los defectos del ciclo de la úrea se deben a deficiencias de diferentes enzimas; las manifestaciones clínicas son similares y están relacionadas con la hiperamonemia. Se presentan las historias clínicas de tres neonatos a término, sin evidencia de alteración al nacimiento. Se les detectó hiperamonemia y se sospechó enfermedad metabólica. La cromatografía de aminoácidos sugirió defectos del ciclo de la úrea. El manejo incluyó dieta con restricción de proteínas, administración de benzoato de sodio, exsanguinotransfusión y diálisis peritoneal pese a lo cual fallecieron. Se revisan las causas de hiperamonemia en el neonato y se propone una secuencia para su diagnósticoThe urea cycle disorders result from deficiency of activity of enzymes N-acetyl glutamate synthetase, carbamyl phosphate synthase, ornithine transcarbamylase, argininosuccinic acid synthetase, argininosuccinic acid lyase and arginase. Except for the last one, the clinical features are similar and related with the hiperammonaemia. It reports three full term, newborn cases, they had encephalopathy and needed respiratory support after be well in neonatal period. They had hyperammonemia as inborn error. The thin layer amino acids chromatography showed alanine and glutamine, in the siblings appeared citruline, suggesting urea cycle disorders. Despite protein restriction diet, sodium benzoate administration, blood exchange and peritoneal dialysis,babies died. High argininosuccinic acid levels in the first case and high citrulline levels with argininosuccinic acid absence in the third case, which was diagnosed as argininosuccinic aciduria with citrullinemia. This report provide an overview of neonatal hyperammonemia causes and propose a secuency for diagnosis

  14. Developmental expression of STATs, nuclear factor-κB and inflammatory genes in the jejunum of piglets during weaning.

    Science.gov (United States)

    Yi, Hongbo; Jiang, Denghu; Zhang, Lin; Xiong, Haitao; Han, Feifei; Wang, Yizhen

    2016-07-01

    The signal transducer and activator of transcription (STAT) proteins play essential roles in apoptosis, proliferation and survival. However, the role of STATs in intestinal inflammation during weaning is unclear. This study aimed to investigate developmental expression of STATs, nuclear factor-κB (NF-κB) and inflammatory genes in the jejunum of piglets during weaning. Thirty-two piglets were weaned at 21d and sacrificed at 0, 1, 7, or 14d (n=8) after weaning. Villus height and the villus height/crypt depth ratio were decreased, whereas crypt depth was increased in the jejunum at 7 and 14d after weaning. In addition, the mRNA levels of interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), IL-6, IL-8, IL-12 and IL-22 were increased in the jejunum at 7 and 14d after weaning, whereas transforming growth factor-β (TGF-β), suppressor of cytokine signaling 3 (SCOS3) and arginase-1 was decreased. Neutrophil infiltration was increased in the mucosa of the jejunum after weaning. Moreover, phosphorylation of IκB-α, NF-κB, AKT and STAT-3 was increased. However, the phosphorylation of STAT-1 (at 7 and 14d) and STAT-6 (at 1 and 7d) was suppressed in the jejunum after weaning. Treatment of porcine jejunal epithelial (IPEC-J2) cells with the STAT inhibitors fludarabine, niclosamide and teriflunomide, which inhibit the phosphorylation of STAT-1, STAT-3 and STAT-6, respectively, weakened the defense capacity of these cells against bacterial infection. In conclusion, weaning caused severe inflammation associated with activation of the NF-κB and STAT-3 pathways and suppression of STAT-1 and STAT-6 in the jejunum of piglets.

  15. Chemerin aggravates DSS-induced colitis by suppressing M2 macrophage polarization.

    Science.gov (United States)

    Lin, Yuli; Yang, Xuguang; Yue, Wenjie; Xu, Xiaofei; Li, Bingji; Zou, Linlin; He, Rui

    2014-07-01

    Chemerin is present in various inflammatory sites and is closely involved in tissue inflammation. Recent studies have demonstrated that chemerin treatment can cause either anti-inflammatory or pro-inflammatory effects according to the disease model being investigated. Elevated circulating chemerin was recently found in patients with inflammatory bowel disease (IBD); however, the role of chemerin in intestinal inflammation remains unknown. In this study, we demonstrated that the administration of exogenous chemerin (aa17-156) aggravated the severity of dextran sulfate sodium (DSS)-induced colitis, which was characterized by higher clinical scores, extensive mucosal damage and significantly increased local and systemic production of pro-inflammatory cytokines, including IL-6, TNF-α and interferon (IFN-γ). Interestingly, chemerin did not appear to influence the magnitudes of inflammatory infiltrates in the colons, but did result in significantly decreased colonic expression of M2 macrophage-associated genes, including Arginase 1 (Arg-1), Ym1, FIZZ1 and IL-10, following DSS exposure, suggesting an impaired M2 macrophage skewing in vivo. Furthermore, an in vitro experiment showed that the addition of chemerin directly suppressed M2 macrophage-associated gene expression and STAT6 phosphorylation in IL-4-stimulated macrophages. Significantly elevated chemerin levels were found in colons from DSS-exposed mice and from ulcerative colitis (UC) patients and appeared to positively correlate with disease severity. Moreover, the in vivo administration of neutralizing anti-chemerin antibody significantly improved intestinal inflammation following DSS exposure. Taken together, our findings reveal a pro-inflammatory role for chemerin in DSS-induced colitis and the ability of chemerin to suppress the anti-inflammatory M2 macrophage response. Our study also suggests that upregulated chemerin in inflamed colons may contribute to the pathogenesis of IBD.

  16. Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation.

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    Ablamunits, Vitaly; Henegariu, Octavian; Hansen, Jakob Bondo; Opare-Addo, Lynn; Preston-Hurlburt, Paula; Santamaria, Pere; Mandrup-Poulsen, Thomas; Herold, Kevan C

    2012-01-01

    Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1β. We postulated that blockade of IL-1β would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1β mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7(+) T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.

  17. Zonation of nitrogen and glucose metabolism gene expression upon acute liver damage in mouse.

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    Shahrouz Ghafoory

    Full Text Available Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl4 injection. CCl4 mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb, arginase (Arg1, glutaminase 2 (Gls2, Glutamine synthetase (Gs, glucose-6-phosphatase (G6pc, glycogen synthase 2 (Gys2, Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh, Cytochrom p450 2E1 (Cyp2e1 and glucagon receptor (Gcgr genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl4 injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl4, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl4 injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery.

  18. Zonation of Nitrogen and Glucose Metabolism Gene Expression upon Acute Liver Damage in Mouse

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    Ghafoory, Shahrouz; Breitkopf-Heinlein, Katja; Li, Qi; Scholl, Catharina; Dooley, Steven; Wölfl, Stefan

    2013-01-01

    Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl4) injection. CCl4 mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb), arginase (Arg1), glutaminase 2 (Gls2), Glutamine synthetase (Gs), glucose-6-phosphatase (G6pc), glycogen synthase 2 (Gys2), Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh), Cytochrom p450 2E1 (Cyp2e1) and glucagon receptor (Gcgr) genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl4 injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl4, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl4 injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery. PMID:24147127

  19. Vasculopathy and pulmonary hypertension in sickle cell disease.

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    Potoka, Karin P; Gladwin, Mark T

    2015-02-15

    Sickle cell disease (SCD) is an autosomal recessive disorder in the gene encoding the β-chain of hemoglobin. Deoxygenation causes the mutant hemoglobin S to polymerize, resulting in rigid, adherent red blood cells that are entrapped in the microcirculation and hemolyze. Cardinal features include severe painful crises and episodic acute lung injury, called acute chest syndrome. This population, with age, develops chronic organ injury, such as chronic kidney disease and pulmonary hypertension. A major risk factor for developing chronic organ injury is hemolytic anemia, which releases red blood cell contents into the circulation. Cell free plasma hemoglobin, heme, and arginase 1 disrupt endothelial function, drive oxidative and inflammatory stress, and have recently been referred to as erythrocyte damage-associated molecular pattern molecules (eDAMPs). Studies suggest that in addition to effects of cell free plasma hemoglobin on scavenging nitric oxide (NO) and generating reactive oxygen species (ROS), heme released from plasma hemoglobin can bind to the toll-like receptor 4 to activate the innate immune system. Persistent intravascular hemolysis over decades leads to chronic vasculopathy, with ∼10% of patients developing pulmonary hypertension. Progressive obstruction of small pulmonary arterioles, increase in pulmonary vascular resistance, decreased cardiac output, and eventual right heart failure causes death in many patients with this complication. This review provides an overview of the pathobiology of hemolysis-mediated endothelial dysfunction and eDAMPs and a summary of our present understanding of diagnosis and management of pulmonary hypertension in sickle cell disease, including a review of recent American Thoracic Society (ATS) consensus guidelines for risk stratification and management.

  20. Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type, which mediates inhibition of tumour immune-evasion via the Toll-like receptor 2 pathway.

    Science.gov (United States)

    Liu, Yi; Zhang, Lingyun; Zhu, Xiangxiang; Wang, Yuehua; Liu, WenWei; Gong, Wei

    2015-11-01

    Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti-cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin-6 (IL-6), IL-12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr-1(+) CD11b(+) monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll-like receptor 2 (TLR2) signal of Gr-1(+) CD11b(+) MDSCs and increased their M1-type macrophage characteristics, such as producing IL-12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr-1(+) CD11b(+) MDSCs by pAbM treatment had less ability to convert the CD4(+) CD25(-) cells into CD4(+) CD25(+) phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti-tumour effect of pAbM was dependent on Gr-1(+ ) CD11b(+) monocytes, nether CD8(+) T cells nor CD4(+) T cells. In addition to, pAbM did not inhibit tumour growth in TLR2(-/-) mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti-tumour effects through the opposite of the suppressive function of Gr-1(+) CD11b(+) MDSCs.

  1. Effects of non-surgical periodontal treatment on the L-arginine-nitric oxide pathway and oxidative status in platelets.

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    Siqueira, Mariana Alves de Sá; Fischer, Ricardo Guimarães; Pereira, Natália Rodrigues; Martins, Marcela Anjos; Moss, Monique Bandeira; Mendes-Ribeiro, Antônio Cláudio; Figueredo, Carlos Marcelo da Silva; Brunini, Tatiana Marlowe Cunha

    2013-06-01

    Several studies have suggested an increase of cardiovascular disease (CVD) risk on periodontitis patients. An enhancement has been demonstrated on both platelet activation and oxidative stress on periodontitis patients, which may contribute for this association. Therefore, the aim of this study was to evaluate the effects of non-surgical periodontal treatment on the l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway and oxidative status in platelets. A total of eight periodontitis patients and eight controls were included in this study. Clinical, laboratory and experimental evaluations were performed on baseline and 90 days after periodontal treatment (except for western blot analysis). The clinical periodontal evaluation included measurements of probing pocket depth (PPD), clinical attachment loss (CAL), % of sites with plaque and % of sites with bleeding on probing. We evaluated: l-[(3)H]arginine influx; nitric oxide synthase (NOS) and arginase enzymes activity and expression; expression of guanylate cyclase and phosphodiesterase-5 enzymes; cGMP levels; platelet aggregation; oxidative status through superoxide dismutase (SOD) and catalase activities, and measurement of reactive oxygen species (ROS) levels and C-reactive protein (CRP) levels. The initial results showed an activation of both l-arginine influx and via system y (+ )L associated with reduced intraplatelet cGMP levels in periodontitis patients and increased systemic levels of CRP. After periodontal treatment, there was a significant reduction of the % of sites with PPD 4-5mm, % of sites with CAL 4-5 mm, and an enhancement in cGMP levels and SOD activity. Moreover, CRP levels were reduced after treatment. Therefore, alterations in the intraplatelet l-arginine-NO-cGMP pathway and oxidant-antioxidant balance associated with a systemic inflammatory response may lead to platelet dysfunction, which may contribute to a higher risk of CVD in periodontitis.

  2. The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

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    Maria Ruweka Fernando

    Full Text Available Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells - immunoregulatory features not observed in the 'parent' IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its' anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

  3. The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

    Science.gov (United States)

    Fernando, Maria Ruweka; Reyes, Jose Luis; Iannuzzi, Jordan; Leung, Gabriella; McKay, Derek Mark

    2014-01-01

    Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells - immunoregulatory features not observed in the 'parent' IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT)-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its' anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

  4. Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic.

    Science.gov (United States)

    Wilkie, Michael P; Turnbull, Steven; Bird, Jonathan; Wang, Yuxiang S; Claude, Jaime F; Youson, John H

    2004-08-01

    We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.

  5. Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

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    van Straten, Giora; van Steenbeek, Frank G; Grinwis, Guy C M; Favier, Robert P; Kummeling, Anne; van Gils, Ingrid H; Fieten, Hille; Groot Koerkamp, Marian J A; Holstege, Frank C P; Rothuizen, Jan; Spee, Bart

    2014-01-01

    The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.

  6. The Chinese soft-shelled turtle, Pelodiscus sinensis, decreases nitrogenous excretion, reduces urea synthesis and suppresses ammonia production during emersion.

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    Ip, Yuen K; Lee, Serene M L; Wong, Wai P; Chew, Shit F

    2013-05-01

    The objective of this study was to examine the effects of 6 days of emersion on nitrogen metabolism and excretion in the Chinese soft-shelled turtle, Pelodiscus sinensis. Despite having a soft shell with a cutaneous surface that is known to be water permeable, P. sinensis lost only ~2% of body mass and was able to maintain its hematocrit and plasma osmolality, [Na(+)] and [Cl(-)] during 6 days of emersion. During emersion, it ameliorated water loss by reducing urine output, which led to a reduction (by 29-76%) in ammonia excretion. In comparison, there was a more prominent reduction (by 82-99%) in urea excretion during emersion due to a lack of water to flush the buccopharyngeal epithelium, which is known to be the major route of urea excretion. Consequently, emersion resulted in an apparent shift from ureotely to ammonotely in P. sinensis. Although urea concentration increased in several tissues, the excess urea accumulated could only account for 13-22% of the deficit in urea excretion. Hence, it can be concluded that a decrease (~80%) in urea synthesis occurred in P. sinensis during the 6 days of emersion. Indeed, emersion led to significant decreases in the activity of some ornithine-urea cycle enzymes (argininosuccinate synthetase/argininosuccinate lyase and arginase) from the liver of P. sinensis. As a decrease in urea synthesis occurred without the accumulation of ammonia and total free amino acids, it can be deduced that ammonia production through amino acid catabolism was suppressed with a proportional reduction in proteolysis in P. sinensis during emersion. Indeed, calculated results revealed that there could be a prominent decrease (~88%) in ammonia production in turtles after 6 days of emersion. In summary, despite being ureogenic and ureotelic in water, P. sinensis adopted a reduction in ammonia production, instead of increased urea synthesis, as the major strategy to ameliorate ammonia toxicity and problems associated with dehydration during

  7. Multiple helminth infection of the skin causes lymphocyte hypo-responsiveness mediated by Th2 conditioning of dermal myeloid cells.

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    Peter C Cook

    2011-03-01

    Full Text Available Infection of the mammalian host by schistosome larvae occurs via the skin, although nothing is known about the development of immune responses to multiple exposures of schistosome larvae, and/or their excretory/secretory (E/S products. Here, we show that multiple (4x exposures, prior to the onset of egg laying by adult worms, modulate the skin immune response and induce CD4(+ cell hypo-responsiveness in the draining lymph node, and even modulate the formation of hepatic egg-induced granulomas. Compared to mice exposed to a single infection (1x, dermal cells from multiply infected mice (4x, were less able to support lymph node cell proliferation. Analysis of dermal cells showed that the most abundant in 4x mice were eosinophils (F4/80(+MHC-II(-, but they did not impact the ability of antigen presenting cells (APC to support lymphocyte proliferation to parasite antigen in vitro. However, two other cell populations from the dermal site of infection appear to have a critical role. The first comprises arginase-1(+, Ym-1(+ alternatively activated macrophage-like cells, and the second are functionally compromised MHC-II(hi cells. Through the administration of exogenous IL-12 to multiply infected mice, we show that these suppressive myeloid cell phenotypes form as a consequence of events in the skin, most notably an enrichment of IL-4 and IL-13, likely resulting from an influx of RELMα-expressing eosinophils. We further illustrate that the development of these suppressive dermal cells is dependent upon IL-4Rα signalling. The development of immune hypo-responsiveness to schistosome larvae and their effect on the subsequent response to the immunopathogenic egg is important in appreciating how immune responses to helminth infections are modulated by repeated exposure to the infective early stages of development.

  8. Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages

    Science.gov (United States)

    Montana, Giovanna

    2016-01-01

    Substance P (SP) is a neuropeptide that mediates many physiological as well as inflammatory responses. Recently, SP has been implicated in the resolution of inflammation through induction of M2 macrophages phenotype. The shift between M1-like and M2-like, allowing the resolution of inflammatory processes, also takes place by means of hemeoxygenase-1 (HO-1). HO-1 is induced in response to oxidative stress and inflammatory stimuli and modulates the immune response through macrophages polarisation. SP induces HO-1 expression in human periodontal ligament (PDL), the latter potentially plays a role in cytoprotection. We demonstrated that SP promotes M2-like phenotype from resting as well as from M1 macrophages. Indeed, SP triggers the production of interleukine-10 (IL-10), interleukine-4 (IL-4) and arginase-1 (Arg1) without nitric oxide (NO) generation. In addition, SP increases HO-1 expression in a dose- and time-dependent manner. Here we report that SP, without affecting cell viability, significantly reduces the production of pro-inflammatory cytokines and enzymes, such as tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and ameliorates migration and phagocytic properties in LPS-stimulated RAW 264.7 cells. M2-like conversion required retention of NF-κB p65 into the cytoplasm and HO-1 induced expression. Silencing of the HO-1 mRNA expression reversed the induction of pro-inflammatory cytokines in RAW 264.7 stimulated by LPS and down-regulated anti-inflammatory hallmarks of M2 phenotype. In conclusion, our data show that SP treatment might be associated with anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression. PMID:27907187

  9. Increased levels of circulating and tumor-infiltrating granulocytic myeloid cells in colorectal cancer patients

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    Salman M Toor

    2016-12-01

    Full Text Available Increased levels of myeloid cells, especially myeloid-derived suppressor cells (MDSCs, have been reported to correlate with bad prognosis and reduced survival in cancer patients. However, limited data are available on their conclusive phenotypes and their correlation with clinical settings. The aim of this study was to investigate levels and phenotype of myeloid cells in peripheral blood and tumor microenvironment of colorectal cancer (CRC patients, compared to blood from healthy donors (HDs and paired, adjacent non-tumor colon tissue. Flow cytometric analysis was performed to examine the expression of different myeloid markers in fresh peripheral blood samples from CRC patients and HDs, and tissue-infiltrating immune cells from CRC patients. We found significantly higher levels of cells expressing myeloid markers and lacking the expression of MHC class II molecule HLA-DR in blood and tumor of CRC patients. Further analysis revealed that these cells were granulocytic and expressed Arginase 1 (ARG1, indicative of their suppressive phenotype. These expanded cells could be neutrophils or granulocytic MDSCs, and we refer to them as granulocytic myeloid cells (GMCs due to the phenotypical and functional overlap between these cell subsets. Interestingly, the expansion of peripheral GMCs correlated with higher stage and histological grade of cancer, thereby suggesting their role in cancer progression. Furthermore, an increase in CD33+CD11b+HLA-DR-CD14-CD15- immature myeloid cells (IMCs was also observed in CRC tumor tissue. Our work shows that GMCs are expanded in circulation and tumor microenvironment of CRC patients, which provides further insights for developing immunotherapeutic approaches targeting these cell subsets to enhance anti-tumor immune and clinical responses.

  10. Renoprotective Mechanism of Remote Ischemic Preconditioning Based on Transcriptomic Analysis in a Porcine Renal Ischemia Reperfusion Injury Model.

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    Young Eun Yoon

    Full Text Available Ischemic preconditioning (IPC is a well-known phenomenon in which tissues are exposed to a brief period of ischemia prior to a longer ischemic event. This technique produces tissue tolerance to ischemia reperfusion injury (IRI. Currently, IPC's mechanism of action is poorly understood. Using a porcine single kidney model, we performed remote IPC with renal IRI and evaluated the IPC mechanism of action. Following left nephrectomy, 15 female Yorkshire pigs were divided into three groups: no IPC and 90 minutes of warm ischemia (control, remote IPC immediately followed by 90 minutes of warm ischemia (rIPCe, and remote IPC with 90 minutes of warm ischemia performed 24 hours later (rIPCl. Differential gene expression analysis was performed using a porcine-specific microarray. The microarray analysis of porcine renal tissues identified 1,053 differentially expressed probes in preconditioned pigs. Among these, 179 genes had altered expression in both the rIPCe and rIPCl groups. The genes were largely related to oxidation reduction, apoptosis, and inflammatory response. In the rIPCl group, an additional 848 genes had altered expression levels. These genes were primarily related to immune response and inflammation, including those coding for cytokines and cytokine receptors and those that play roles in the complement system and coagulation cascade. In the complement system, the membrane attack complex was determined to be sublytic, because it colocalized with phosphorylated extracellular signal-regulated kinase. Furthermore, alpha 2 macroglobulin, tissue plasminogen activator, uterine plasmin trypsin inhibitor, and arginase-1 mRNA levels were elevated in the rIPCl group. These findings indicate that remote IPC produces renoprotective effects through multiple mechanisms, and these effects develop over a long timeframe rather than immediately following IPC.

  11. Feeding low or pharmacological concentrations of zinc oxide changes the hepatic proteome profiles in weaned piglets.

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    Angelika Bondzio

    Full Text Available Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter. After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05 expressed between groups receiving control (150 mg/kg or pharmacological levels of zinc (2500 mg/kg with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70 and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs.

  12. Cerebral and peripheral changes occurring in nitric oxide (NO synthesis in a rat model of sleeping sickness: identification of brain iNOS expressing cells.

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    Donia Amrouni

    Full Text Available BACKGROUND: The implication of nitric oxide (NO in the development of human African trypanosomiasis (HAT using an animal model, was examined. The manner by which the trypanocidal activity of NO is impaired in the periphery and in the brain of rats infected with Trypanosoma brucei brucei (T. b. brucei was analyzed through: (i the changes occurring in NO concentration in both peripheral (blood and cerebral compartments; (ii the activity of nNOS and iNOS enzymes; (iii identification of the brain cell types in which the NO-pathways are particularly active during the time-course of the infection. METHODOLOGY/PRINCIPAL FINDINGS: NO concentration (direct measures by voltammetry was determined in central (brain and peripheral (blood compartments in healthy and infected animals at various days post-infection: D5, D10, D16 and D22. Opposite changes were observed in the two compartments. NO production increased in the brain (hypothalamus from D10 (+32% to D16 (+71%, but decreased in the blood from D10 (-22% to D16 (-46% and D22 (-60%. In parallel with NO measures, cerebral iNOS activity increased and peaked significantly at D16 (up to +700%. However, nNOS activity did not vary. Immunohistochemical staining confirmed iNOS activation in several brain regions, particularly in the hypothalamus. In peritoneal macrophages, iNOS activity decreased from D10 (-83% to D16 (-65% and D22 (-74% similarly to circulating NO. CONCLUSION/SIGNIFICANCE: The NO changes observed in our rat model were dependent on iNOS activity in both peripheral and central compartments. In the periphery, the NO production decrease may reflect an arginase-mediated synthesis of polyamines necessary to trypanosome growth. In the brain, the increased NO concentration may result from an enhanced activity of iNOS present in neurons and glial cells. It may be regarded as a marker of deleterious inflammatory reactions.

  13. Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

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    Patassini, Stefano; Begley, Paul; Reid, Suzanne J; Xu, Jingshu; Church, Stephanie J; Curtis, Maurice; Dragunow, Mike; Waldvogel, Henry J; Unwin, Richard D; Snell, Russell G; Faull, Richard L M; Cooper, Garth J S

    Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

  14. A Coral-Derived Compound Improves Functional Recovery after Spinal Cord Injury through Its Antiapoptotic and Anti-Inflammatory Effects

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    Chun-Hong Chen

    2016-09-01

    Full Text Available Background: Our previous in vitro results demonstrated that 11-dehydrosinulariolide significantly reduced 6-hydroxydopamine-induced cytotoxicity and apoptosis in a human neuroblastoma cell line, SH-SY5Y, and suppressed the expression of inducible NO synthase (iNOS and cyclooxygenase 2 in lipopolysaccharide-stimulated macrophage cells. The neuroprotective and anti-inflammatory effects of 11-dehydrosinulariolide may be suitable for treating spinal cord injury (SCI. Methods: In the present study, Wistar rats were pretreated with 11-dehydrosinulariolide or saline through intrathecal injection after a thoracic spinal cord contusion injury induced using a New York University (NYU impactor. The apoptotic cells were assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. The expression and localization of proinflammatory, apoptosis-associated and cell survival-related pathway proteins were examined through immunoblotting and immunohistochemistry. Results: 11-Dehydrosinulariolide attenuated SCI-induced cell apoptosis by upregulating the antiapoptotic protein Bcl-2 and cell survival-related pathway proteins p-Akt and p-ERK, 8 h after SCI. Furthermore, the transcription factor p-CREB, which regulates Bcl-2 expression, was upregulated after 11-dehydrosinulariolide treatment. On day 7 after SCI, 11-dehydrosinulariolide exhibited an anti-inflammatory effect, attenuating SCI-induced upregulation of the inflammatory proteins iNOS and tumor necrosis factor-α. 11-Dehydrosinulariolide also induced an increase in the expression of arginase-1 and CD206, markers of M2 microglia, in the injured spinal cord on day 7 after SCI. Thus, the anti-inflammatory effect of 11-dehydrosinulariolide may be related to the promotion of an alternative pathway of microglia activation. Conclusion: The results show that 11-dehydrosinulariolide exerts antiapoptotic effects at 8 h after SCI and anti-inflammatory effects at 7 days after SCI. We

  15. Effects of tamoxifen on vaginal blood flow and epithelial morphology in the rat

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    Goldstein Irwin

    2006-09-01

    Full Text Available Abstract Background Tamoxifen, a selective estrogen receptor modulator with both estrogenic and anti-estrogenic activity, is widely used as adjuvant therapy in breast cancer patients. Treatment with tamoxifen is associated with sexual side effects, such as increased vaginal dryness and pain/discomfort during sexual activity. There have been limited investigations of the effect of tamoxifen on estrogen-dependent peripheral genital arousal responses. The objective of this study was to investigate the effects of tamoxifen on vaginal physiology in the rat. Methods Female Sprague-Dawley rats were subjected to sham surgery or bilateral ovariectomy. After 2 weeks, sham-operated rats were implanted with subcutaneous osmotic infusion pumps containing vehicle (control or tamoxifen (150 μg/day. Ovariectomized rats were similarly infused with vehicle. After an additional 2 weeks, vaginal blood flow responses to pelvic nerve stimulation were measured by laser Doppler flowmetry and vaginal tissue was collected for histological and biochemical assay. Results Tamoxifen treatment did not change plasma estradiol concentrations relative to control animals, while ovariectomized rats exhibited a 60% decrease in plasma estradiol. Tamoxifen treatment caused a significant decrease in mean uterine weight, but did not alter mean vaginal weight. Vaginal blood flow was significantly decreased in tamoxifen-infused rats compared to controls. Similar to ovariectomized animals, estrogen receptor binding was increased and arginase enzyme activity was decreased in tamoxifen-infused rats. However, different from control and ovariectomized animals, the vaginal epithelium in tamoxifen-infused rats appeared highly mucified. Periodic acid-Schiff staining confirmed a greater production of carbohydrate-rich compounds (e.g. mucin, glycogen by the vaginal epithelium of tamoxifen-infused rats. Conclusion The observations suggest that tamoxifen exerts both anti-estrogenic and pro

  16. The role of myeloid-derived suppressor cells in immune ontogeny

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    Soren eGantt

    2014-08-01

    Full Text Available Myeloid derived suppressor cells (MDSC are a heterogeneous population of granulocytic or monocytic cells that suppress innate as well as adaptive immune responses. In healthy adults, immature myeloid cells differentiate into macrophages, dendritic cells, and granulocytes in the bone marrow, and MDSC are rarely detected in peripheral blood. However, in certain pathologies, in particular malignancies and chronic infection, differentiation of these cells is altered resulting in accumulation of circulating suppressive myeloid cells. MDSC express suppressive factors such as arginase-1, reactive oxygen species, and inducible nitric oxide synthase, which have the ability to inhibit T cell proliferation and cytoxicity, induce the expansion of regulatory T cells, and block natural killer cell activation. It is increasingly recognized that MDSC alter the immune response to several cancers, and perhaps chronic viral infections, in clinically important ways. In this review, we outline the potential contribution of MDSC to the generation of feto-maternal tolerance and to the ineffective immune responses to many infections and vaccines observed in early post-natal life. Granulocytic MDSC are present in large numbers in pregnant women and in cord blood, and wane rapidly during infancy. Furthermore, cord blood MDSC suppress in vitro T cell and NK responses, suggesting that they may play a significant role in human immune ontogeny. However, there are currently no data that demonstrate in vivo effects of MDSC on feto-maternal tolerance or immune ontogeny. Studies are ongoing to evaluate the functional importance of MDSC, including their effects on control of infection and response to vaccination in infancy. Importantly, several pharmacologic interventions have the potential to reverse MDSC function. Understanding the role of MDSC in infant ontogeny and their mechanisms of action could lead to interventions that reduce mortality due to early-life infections.

  17. Targeting polyamine metabolism for finding new drugs against leishmaniasis: a review.

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    Ilari, Andrea; Fiorillo, Annarita; Baiocco, Paola; Poser, Elena; Angiulli, Gabriella; Colotti, Gianni

    2015-01-01

    Leishmaniasis is a neglected disease affecting more than 12 million people worldwide. The most used drugs are pentavalent antimonials that are very toxic and display the problem of drug resistance, especially in endemic regions such as Bihar in India. For this reason, it is urgent to find new and less toxic drugs against leishmaniasis. To this end, the understanding of pathways affecting parasite survival is of prime importance for targeted drug discovery. The parasite survival inside the macrophage is strongly dependent on polyamine metabolism. Polyamines are, in fact, very important for cell growth and proliferation. In particular, spermidine (Spd), the final product of the polyamine biosynthesis pathway, serves as a precursor for trypanothione (N1,N8- bis(glutathionyl)spermidine, T(SH)2) and hypusine (N(ε)-(4-amino-2-hydroxybutyl)lysine). T(SH)2 is a key molecule for parasite defense against the hydrogen peroxide produced by macrophages during the infection. Hypusination is a posttranslational modification occurring exclusively in the eukaryotic initiation factor 5A (eIF5A), which has an important role in avoiding the ribosome stalling during the biosynthesis of protein containing polyprolines sequences. The enzymes, belonging to the spermidine metabolism, i.e. arginase (ARG), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase (SpdS), trypanothione synthetase (TryS or TSA), trypanothione reductase (TryR or TR), tryparedoxin peroxidase (TXNPx), deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are promising targets for the development of new drugs against leishmaniasis. This minireview furnishes a picture of the structural, functional and inhibition studies on polyamine metabolism enzymes that could guide the discovery of new drugs against leishmaniasis.

  18. Induction of microglia activation after infection with the non-neurotropic A/CA/04/2009 H1N1 influenza virus.

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    Shankar Sadasivan

    Full Text Available Although influenza is primarily a respiratory disease, it has been shown, in some cases, to induce encephalitis, including people acutely infected with the pandemic A/California/04/2009 (CA/09 H1N1 virus. Based on previous studies showing that the highly pathogenic avian influenza (HPAI A/Vietnam/1203/2004 H5N1 virus was neurotropic, induced CNS inflammation and a transient parkinsonism, we examined the neurotropic and inflammatory potential of the CA/09 H1N1 virus in mice. Following intranasal inoculation, we found no evidence for CA/09 H1N1 virus neurotropism in the enteric, peripheral or central nervous systems. We did, however, observe a robust increase in microglial activity in the brain characterized by an increase in the number of activated Iba-1-positive microglia in the substantia nigra (SN and the hippocampus, despite the absence of virus in the brain. qPCR analysis in SN tissue showed that the induction of microgliosis was preceded by reduced gene expression of the neurotrophic factors bdnf, and gdnf and increases in the immune modulatory chemokine chemokine (C-C motif ligand 4 (ccl4. We also noted changes in the expression of transforming growth factor-1 (tgfβ1 in the SN starting at 7 days post-infection (dpi that was sustained through 21 dpi, coupled with increases in arginase-1 (arg1 and csf1, M2 markers for microglia. Given that neuroinflammation contributes to generation and progression of a number of neurodegenerative disorders, these findings have significant implications as they highlight the possibility that influenza and perhaps other non-neurotropic viruses can initiate inflammatory signals via microglia activation in the brain and contribute to, but not necessarily be the primary cause of, neurodegenerative disorders.

  19. [Biochemical mechanisms of the cardioprotective effect of the K(ATP) channels opener flocalin (medicinal form) in ischemia-reperfusion of myocardium].

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    Strutyns'kyĭ, R B; Kotsiuruba, A V; Rovenets', R A; Strutyns'ka, N A; Iagupols'kyĭ, Iu L; Sagach, V F; Moĭbenko, O O

    2013-01-01

    In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) of myocardium it was investigated changes of biochemical processes in arterial blood at intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible mechanisms of cardioprotective action offlocalin, which prevented the opening of a mitochondrial permeability transition pore (MPTP) and inhibition of apoptosis induced by it. They consist, from one side, in activating of the constitutive de novo biosynthesis of nitric oxide by cNOS, from other side, in suppression of inducible nitric oxide de novo synthesis by iNOS in such way to prevent the formation of toxic peroxynitrite by co-operation of surplus nitric oxide with superoxide anion, thereby limits the generation of toxic active forms of nitrogen (*NO2) and oxygen (*OH). The first effect of flocalin takes place due to limitation the degradation of L-arginine by arginase which keeps substrat for cNOS, second--due to the inhibition of superoxide generation, in particular, by xanthine oxidase (marker uric acid), lipoxigenase (marker LTC4) and cyclooxygenase (marker TxB2). Because LTC4 have coronaroconstrictory, arrhythmogenic and chemoattractory properties in the conditions of myocardial ischemia, inhibition of its production both with superoxide generation (markers H2O2 and diene conjugates) may be the another mechanisms of flocalin's cardioprotection. Powerful antiischemic action of flocalin (marker nitrite anion) as the mechanisms of cardioprotection is possible as well as inhibition of ATP and GTP degradation (marker hypoxanthine+xanthine+inosine levels in the blood) and, possibly, stimulation ofhaem degradation by haem oxygenase (markers total bilirubin and Fe in the blood). Diminishing content of free arachidonic acid in arterial blood can testify

  20. The role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases

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    Kristina S. Burrack

    2014-09-01

    Full Text Available When an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. Myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase 2 (Nos2; iNOS and arginase 1 (Arg1. Nitric oxide (NO production by iNOS is an important proinflammatory mediator, whereas Arg1-expressing macrophages contribute to the resolution of inflammation and wound repair. In the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. NO has direct antiviral properties against some viruses, whereas during other virus infections NO can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. Arg1 activity has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. Thus, depending on the specific virus and the tissue(s involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. In this review, we will discuss a variety of viral infections, including HIV, SARS-CoV, LCMV, HCV, RSV, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of iNOS and/or Arg1. Clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections.

  1. Interleukin-17 induces an atypical M2-like macrophage subpopulation that regulates intestinal inflammation.

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    Kenichiro Nishikawa

    Full Text Available Interleukin 17 (IL-17 is a pleiotropic cytokine that acts on both immune and non-immune cells and is generally implicated in inflammatory and autoimmune diseases. Although IL-17 as well as their source, mainly but not limited to Th17 cells, is also abundant in the inflamed intestine, the role of IL-17 in inflammatory bowel disease remains controversial. In the present study, by using IL-17 knockout (KO mice, we investigated the role of IL-17 in colitis, with special focus on the macrophage subpopulations. Here we show that IL-17KO mice had increased susceptibility to DSS-induced colitis which was associated with decrease in expression of mRNAs implicated in M2 and/or wound healing macrophages, such as IL-10, IL-1 receptor antagonist, arginase 1, cyclooxygenase 2, and indoleamine 2,3-dioxygenase. Lamina propria leukocytes from inflamed colon of IL-17KO mice contained fewer CD11b+Ly6C+MHC Class II+ macrophages, which were derived, at least partly, from blood monocytes, as compared to those of WT mice. FACS-purified CD11b+ cells from WT mice, which were more abundant in Ly6C+MHC Class II+ cells, expressed increased levels of genes associated M2/wound healing macrophages and also M1/proinflammatory macrophages. Depletion of this population by topical administration of clodronate-liposome in the colon of WT mice resulted in the exacerbation of colitis. These results demonstrate that IL-17 confers protection against the development of severe colitis through the induction of an atypical M2-like macrophage subpopulation. Our findings reveal a previously unappreciated mechanism by which IL-17 exerts a protective function in colitis.

  2. Proinflammatory-activated glioma cells induce a switch in microglial polarization and activation status, from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells

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    Lucia Lisi

    2014-05-01

    Full Text Available Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b, with up-regulation of iNOS (inducible nitric oxide synthase, ARG (arginase and IL (interleukine-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide–IFNγ (interferon γ conditioned media] and C-CM (control-conditioned media induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well.

  3. Female immune system is protected from effects of prenatal exposure to mercury

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    Kayla L. Penta

    2015-06-01

    Full Text Available Mercury is a ubiquitous environmental toxicant which bioaccumulates and has many biological effects, including detrimental effects on the nervous and immune systems. Because mercury can cross the placenta and concentrates in the fetal compartment, the developing fetus is particularly vulnerable. We hypothesize that developmental exposure to mercury will cause immunological changes, leading to an increased susceptibility to, or exacerbation of, immune disorders later in life. To better understand these changes, we exposed pregnant female mice to low doses of mercury for a short duration and examined the genetic effects related to immune function in the adult offspring. Pregnant BALB/c mice were exposed to mercury (200 µg/kg HgCl2 in PBS by subcutaneous injection or vehicle control every other day from gestation day 5 to 15. Offspring remained with the dam until weaning and were euthanized at 8 weeks of age with no further exposures to mercury. Splenic RNA was isolated and gene expression changes examined by microarray in a non-random subset of samples and changes confirmed by quantitative PCR. Epigenetic changes were also examined in terms of miRNA levels in the spleen. Although male and female offspring were exposed to mercury in the same in utero environment, the effects on expression of immune-related genes and immune-regulatory epigenetic signals were different dependent upon the sex of the offspring with males, but not females, displaying up-regulation at least two-fold of arginase, interferon-γ, STAT1, vitronectin, and TNFSF18. Epigenetic changes in miRNA levels were differentially expressed in males and females with in utero mercury exposure; miR-191-5p was decreased in males, while miR-1188-3p was increased in females. These gene expression and gene regulation changes modulate the baseline immune response and may impact risks for autoimmunity later in life.

  4. Adipose tissue macrophages in non-rodent mammals: a comparative study.

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    Ampem, Grace; Azegrouz, Hind; Bacsadi, Árpád; Balogh, Lajos; Schmidt, Susanne; Thuróczy, Julianna; Röszer, Tamás

    2016-02-01

    The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.

  5. Identification of a fatty acid binding protein4-UCP2 axis regulating microglial mediated neuroinflammation.

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    Duffy, Cayla M; Xu, Hongliang; Nixon, Joshua P; Bernlohr, David A; Butterick, Tammy A

    2017-02-16

    Hypothalamic inflammation contributes to metabolic dysregulation and the onset of obesity. Dietary saturated fats activate microglia via a nuclear factor-kappa B (NFκB) mediated pathway to release pro-inflammatory cytokines resulting in dysfunction or death of surrounding neurons. Fatty acid binding proteins (FABPs) are lipid chaperones regulating metabolic and inflammatory pathways in response to fatty acids. Loss of FABP4 in peripheral macrophages via either molecular or pharmacologic mechanisms results in reduced obesity-induced inflammation via a UCP2-redox based mechanism. Despite the widespread appreciation for the role of FABP4 in mediating peripheral inflammation, the expression of FABP4 and a potential FABP4-UCP2 axis regulating microglial inflammatory capacity is largely uncharacterized. To that end, we hypothesized that microglial cells express FABP4 and that inhibition would upregulate UCP2 and attenuate palmitic acid (PA)-induced pro-inflammatory response. Gene expression confirmed expression of FABP4 in brain tissue lysate from C57Bl/6J mice and BV2 microglia. Treatment of microglial cells with an FABP inhibitor (HTS01037) increased expression of Ucp2 and arginase in the presence or absence of PA. Moreover, cells exposed to HTS01037 exhibited attenuated expression of inducible nitric oxide synthase (iNOS) compared to PA alone indicating reduced NFκB signaling. Hypothalamic tissue from mice lacking FABP4 exhibit increased UCP2 expression and reduced iNOS, tumor necrosis factor-alpha (TNF-α), and ionized calcium-binding adapter molecule 1 (Iba1; microglial activation marker) expression compared to wild type mice. Further, this effect is negated in microglia lacking UCP2, indicating the FABP4-UCP2 axis is pivotal in obesity induced neuroinflammation. To our knowledge, this is the first report demonstrating a FABP4-UCP2 axis with the potential to modulate the microglial inflammatory response.

  6. Iron-dependent remodeling of fungal metabolic pathways associated with ferrichrome biosynthesis.

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    Mercier, Alexandre; Labbé, Simon

    2010-06-01

    The fission yeast Schizosaccharomyces pombe excretes and accumulates the hydroxamate-type siderophore ferrichrome. The sib1(+) and sib2(+) genes encode, respectively, a siderophore synthetase and an l-ornithine N(5)-oxygenase that participate in ferrichrome biosynthesis. In the present report, we demonstrate that sib1(+) and sib2(+) are repressed by the GATA-type transcriptional repressor Fep1 in response to high levels of iron. We further found that the loss of Fep1 results in increased ferrichrome production. We showed that a sib1Delta sib2Delta mutant strain exhibits a severe growth defect on iron-poor media. We determined that two metabolic pathways are involved in biosynthesis of ornithine, an obligatory precursor of ferrichrome. Ornithine is produced by hydrolysis of arginine by the Car1 and Car3 proteins. Although car3(+) was constitutively expressed, car1(+) transcription levels were repressed upon exposure to iron, with a concomitant decrease of Car1 arginase activity. Ornithine is also generated by transformation of glutamate, which itself is produced by two separate biosynthetic pathways which are transcriptionally regulated by iron in an opposite fashion. In one pathway, the glutamate dehydrogenase Gdh1, which produces glutamate from 2-ketoglutarate, was repressed under iron-replete conditions in a Fep1-dependent manner. The other pathway involves two coupled enzymes, glutamine synthetase Gln1 and Fe-S cluster-containing glutamate synthase Glt1, which were both repressed under iron-limiting conditions but were expressed under iron-replete conditions. Collectively, these results indicate that under conditions of iron deprivation, yeast remodels metabolic pathways linked to ferrichrome synthesis in order to limit iron utilization without compromising siderophore production and its ability to sequester iron from the environment.

  7. Plasmin and Plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via Annexin A1.

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    Sugimoto, Michelle A; Ribeiro, Ana Luíza C; Costa, Bruno R C; Vago, Juliana P; Lima, Kátia M; Carneiro, Fernanda S; Ortiz, Mylena Maira O; Lima, Graziele Letícia N; Carmo, Aline A F; Rocha, Renata M; Perez, Denise A; Reis, Alessandra C; Pinho, Vanessa; Miles, Lindsey A; Garcia, Cristiana C; Teixeira, Mauro M; Sousa, Lirlândia P

    2017-03-20

    Inflammation resolution is an active process that functions to restore tissue homeostasis. Participation of the plasminogen/plasmin (Plg/Pla) system in the productive phase of inflammation is well known, but its involvement in the resolution phase remains unclear. Therefore, we aimed to investigate the potential role of Plg/Pla in key events during the resolution of acute inflammation and its underlying mechanisms. Plg/Pla injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that were primarily macrophages of anti-inflammatory (M2 - F4/80(high) Gr1(-) CD11b(high)) and proresolving (Mres - F4/80(med) CD11b(low)) phenotypes, without changing the number of macrophages with a pro-inflammatory profile (M1 - F4/80(low) Gr1(+) CD11b(med)). Pleural injection of Plg/Pla also increased M2 markers (CD206 and Arginase-1) and secretory products (TGF-β and IL-6) and decreased the expression of iNOS (M1 marker). During the resolving phase of LPS-induced inflammation, when resolving macrophages predominate, we found increased Plg expression and Pla activity, further supporting a link between the Plg/Pla system and key cellular events in resolution. Indeed, Plg or Pla given at the peak of inflammation promoted resolution by decreasing neutrophil numbers and increasing neutrophil apoptosis and efferocytosis in a serine-protease inhibitor sensitive manner. Next, we confirmed the ability of Plg/Pla to both promote efferocytosis and override the pro-survival effect of LPS, via AnxA1. These findings suggest that Plg/Pla regulate several key steps in inflammation resolution, namely, neutrophil apoptosis, macrophage reprogramming and efferocytosis, which have a major impact on the establishment of an efficient resolution process.

  8. Targeting colon cancer cell NF-κB promotes an anti-tumour M1-like macrophage phenotype and inhibits peritoneal metastasis.

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    Ryan, A E; Colleran, A; O'Gorman, A; O'Flynn, L; Pindjacova, J; Lohan, P; O'Malley, G; Nosov, M; Mureau, C; Egan, L J

    2015-03-19

    In a model of peritoneal metastasis in immune-competent mice, we show that nuclear factor (NF)-κB inhibition in CT26 colon cancer cells prevents metastasis. NF-κB inhibition, by stable overexpression of IκB-α super-repressor, induced differential polarization of co-cultured macrophages to an M1-like anti-tumour phenotype in vitro. NF-κB-deficient cancer cell-conditioned media (CT26/IκB-α SR) induced interleukin (IL)-12 and nitric oxide (NO) synthase (inducible NO synthase (iNOS)) expression in macrophages. Control cell (CT26/EV) conditioned media induced high levels of IL-10 and arginase in macrophages. In vivo, this effect translated to reduction in metastasis in mice injected with CT26/ IκB-α SR cells and was positively associated with increased CD8(+)CD44(+)CD62L(-) and CD4(+)CD44(+)CD62L(-) effector T cells. Furthermore, inhibition of NF-κB activity induced high levels of NO in infiltrating immune cells and decreases in matrix metalloproteinase-9 expression, simultaneous with increases in tissue inhibitor of metalloproteinases 1 and 2 within tumours. CT26/IκB-α SR tumours displayed increased pro-inflammatory gene expression, low levels of angiogenesis and extensive intratumoral apoptosis, consistent with the presence of an anti-tumour macrophage phenotype. Macrophage depletion reduced tumour size in CT26/EV-injected animals and increased tumour size in CT26/IκB-α SR cells compared with untreated tumours. Our data demonstrate, for the first time, that an important implication of targeting tumour cell NF-κB is skewing of macrophage polarization to an anti-tumour phenotype. This knowledge offers novel therapeutic opportunities for anticancer treatment.

  9. Effects of inhaled L-arginine administration in a murine model of acute asthma.

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    Zeynep Arikan-Ayyildiz

    2014-10-01

    Full Text Available Increased arginase activity in the airways decreases L-arginine and causes deficiency of bronchodilating and anti-inflammatory nitric oxide (NO in asthma. As, it is suggested that L-arginine may have therapeutic potential in asthma treatment, we aimed to investigate the effects of inhaled L-arginine on oxygen saturation (SaO₂ and airway histology in a murine model of acute asthma. Twenty eight BALB/c mice were divided into four groups; I, II, III and IV (control. All groups except the control were sensitized and challenged with ovalbumin. After establishement of acute asthma attack by metacholine administration, the mice were treated with inhaled L-arginine (Group I, saline (Group II and budesonide (Group III, respectively. SaO₂was measured by pulse oximeter just before and 5 min after methacholine. A third measurement of SaO₂was also obtained 15 min after drug administration in these study groups. Inflammation in the lung tissues of the sacrificed animals were scored to determine the effects of the study drugs. The number of eosinophils in bronchoalveolar lavage (BAL was determined. The results indicated that inflammatory scores significantly improved in groups receiving study drugs when compared with placebo and L-arginine was similar in decreasing scores when compared with budesonide. SaO₂had a tendency to increase after L-arginine administration after acute asthma attack and this increase was statistically significant (p=0.043. Eosinophilia in BAL significantly reduced in group receiving L-arginine when compared with placebo (p<0.05. Thus in this study we demonstrated that L-arginine improved SaO₂and inflammatory scores in an acute model of asthma.

  10. Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

    Science.gov (United States)

    Hühner, Laura; Rilka, Jennifer; Gilsbach, Ralf; Zhou, Xiaolai; Machado, Venissa; Spittau, Björn

    2017-01-01

    Microglia are involved in physiological as well as neuropathological processes in the central nervous system (CNS). Their functional states are often referred to as M1-like and M2-like activation, and are believed to contribute to neuroinflammation-mediated neurodegeneration or neuroprotection, respectively. Parkinson’s disease (PD) is one the most common neurodegenerative disease and is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra resulting in bradykinesia, tremor, and rigidity. Interleukin 4 (IL4)-mediated M2-like activation of microglia, which is characterized by upregulation of alternative markers Arginase 1 (Arg1) and Chitinase 3 like 3 (Ym1) has been well studied in vitro but the role of endogenous IL4 during CNS pathologies in vivo is not well understood. Interestingly, microglia activation by IL4 has been described to promote neuroprotective and neurorestorative effects, which might be important to slow the progression of neurodegenerative diseases. In the present study, we addressed the role of endogenous and exogenous IL4 during MPP+-induced degeneration of mDA neurons in vitro and further addressed the impact of IL4-deficiency on neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Our results clearly demonstrate that exogenous IL4 is important to protect mDA neurons in vitro, but endogenous IL4 seems to be dispensable for development and maintenance of the nigrostriatal system as well as MPTP-induced loss of TH+ neurons in vivo. These results underline the importance of IL4 in promoting a neuroprotective microglia activation state and strengthen the therapeutic potential of exogenous IL4 for protection of mDA neurons in PD models. PMID:28337124

  11. Fluorescent imaging of antigen released by a skin-invading helminth reveals differential uptake and activation profiles by antigen presenting cells.

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    Ross A Paveley

    Full Text Available Infection of the mammalian host by the parasitic helminth Schistosoma mansoni is accompanied by the release of excretory/secretory molecules (ES from cercariae which aid penetration of the skin. These ES molecules are potent stimulants of innate immune cells leading to activation of acquired immunity. At present however, it is not known which cells take up parasite antigen, nor its intracellular fate. Here, we develop a technique to label live infectious cercariae which permits the imaging of released antigens into macrophages (MPhi and dendritic cells (DCs both in vitro and in vivo. The amine reactive tracer CFDA-SE was used to efficiently label the acetabular gland contents of cercariae which are released upon skin penetration. These ES products, termed '0-3hRP', were phagocytosed by MHC-II(+ cells in a Ca(+ and actin-dependent manner. Imaging of a labelled cercaria as it penetrates the host skin over 2 hours reveals the progressive release of ES material. Recovery of cells from the skin shows that CFDA-SE labelled ES was initially (3 hrs taken up by Gr1(+MHC-II(- neutrophils, followed (24 hrs by skin-derived F4/80(+MHC-II(lo MPhi and CD11c(+ MHC-II(hi DC. Subsequently (48 hrs, MPhi and DC positive for CFDA-SE were detected in the skin-draining lymph nodes reflecting the time taken for antigen-laden cells to reach sites of immune priming. Comparison of in vitro-derived MPhi and DC revealed that MPhi were slower to process 0-3hRP, released higher quantities of IL-10, and expressed a greater quantity of arginase-1 transcript. Combined, our observations on differential uptake of cercarial ES by MPhi and DC suggest the development of a dynamic but ultimately balanced response that can be potentially pushed towards immune priming (via DC or immune regulation (via MPhi.

  12. Paracoccin Induces M1 Polarization of Macrophages via Interaction with TLR4

    Science.gov (United States)

    Freitas, Mateus S.; Oliveira, Aline F.; da Silva, Thiago A.; Fernandes, Fabrício F.; Gonçales, Relber A.; Almeida, Fausto; Roque-Barreira, Maria C.

    2016-01-01

    The fungal human pathogen Paracoccidioides brasiliensis contains paracoccin (PCN), a multi-domain protein that has lectin and N-acetyl-glucosaminidase activities, which account for its effects on the growth and morphogenesis of the fungus and on the activation of host macrophages through its interaction with TLR N-glycans. With the purpose of detailing the knowledge on the effects of PCN on macrophages, we used recombinant PCN expressed in Pichia pastoris (p-rPCN) to stimulate isolated murine peritoneal macrophages. The activation of these cells manifested through the release of high levels of inflammatory mediators, such as nitric oxide, TNF-α, IL-12p40, and IL-6. Furthermore, peritoneal macrophages stimulated with p-rPCN increased the relative expression of STAT1, SOCS3, and iNOS2 mRNA (M1 polarization markers). However, the expression of Arginase-1, Ym-1, and FIZZ1 (M2 polarization markers) remained at basal levels. Interestingly, the observed M1 macrophages’ polarization triggered by p-rPCN was abolished in cells obtained from knockout Toll-like receptor-4 mice. In this case, the p-rPCN-induced production of pro-inflammatory mediators was blocked too. These results demonstrate that the classical activation of macrophages induced by paracoccin depends on TLR4. Taken together, the results of our study indicate that paracoccin acts as a TLR agonist able to modulate immunity and exerts biological activities that favor its applicability as an immunotherapeutic agent to combat systemic fungal infections. PMID:27458431

  13. Feeding low or pharmacological concentrations of zinc oxide changes the hepatic proteome profiles in weaned piglets.

    Science.gov (United States)

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs.

  14. Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

    Science.gov (United States)

    Louveau, Antoine; Nerrière-Daguin, Véronique; Vanhove, Bernard; Naveilhan, Philippe; Neunlist, Michel; Nicot, Arnaud; Boudin, Hélène

    2015-12-01

    Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.

  15. Autophagic flux regulates microglial phenotype according to the time of oxygen-glucose deprivation/reperfusion.

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    Xia, Cong-Yuan; Zhang, Shuai; Chu, Shi-Feng; Wang, Zhen-Zhen; Song, Xiu-Yun; Zuo, Wei; Gao, Yan; Yang, Peng-Fei; Chen, Nai-Hong

    2016-10-01

    Microglial phenotype alternation is a potential novel pathogenic mechanism for cerebral ischemia. Cerebral ischemia induced autophagy aggravates inflammation and neural injury. However, the effect of autophagy in the modulation of microglial phenotype is still unknown. In this study, we investigated the role of autophagic flux in the alternation of microglial phenotype following oxygen glucose deprivation/reperfusion (OGD/R) in BV-2 cells. Inhibition of autophagic flux by NH4Cl exposure significantly increased the level of microtubule-associated protein 1 light chain 3 (LC3)-II and p62 in control and OGD/R (12h, 24h and 48h) groups, but did not change their expression in OGD/R 72h group, indicating that autophagic flux was inhibited at OGD/R 72h. Once autophagic flux was inhibited at OGD/R 72h or at OGD/R 24h (with NH4Cl), BV-2 cells mainly showed M1 phenotype with increased tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and decreased M2 markers including interleukin-10 (IL-10), Arginase 1 (Arg-1), and brain derived neurotrophic factor (BDNF). Further study indicated that inhibition of autophagic flux activated NF-κB pathway and decreased the activity of cAMP-response element binding protein (CREB), which contributed to the alternation of microglial phenotype. Therefore, inhibition of autophagic flux regulated the alternation of microglial phenotype by modulating the balance between NF-κB and CREB.

  16. Enriched environment decreases microglia and brain macrophages inflammatory phenotypes through adiponectin-dependent mechanisms: Relevance to depressive-like behavior.

    Science.gov (United States)

    Chabry, Joëlle; Nicolas, Sarah; Cazareth, Julie; Murris, Emilie; Guyon, Alice; Glaichenhaus, Nicolas; Heurteaux, Catherine; Petit-Paitel, Agnès

    2015-11-01

    Regulation of neuroinflammation by glial cells plays a major role in the pathophysiology of major depression. While astrocyte involvement has been well described, the role of microglia is still elusive. Recently, we have shown that Adiponectin (ApN) plays a crucial role in the anxiolytic/antidepressant neurogenesis-independent effects of enriched environment (EE) in mice; however its mechanisms of action within the brain remain unknown. Here, we show that in a murine model of depression induced by chronic corticosterone administration, the hippocampus and the hypothalamus display increased levels of inflammatory cytokines mRNA, which is reversed by EE housing. By combining flow cytometry, cell sorting and q-PCR, we show that microglia from depressive-like mice adopt a pro-inflammatory phenotype characterized by higher expression levels of IL-1β, IL-6, TNF-α and IκB-α mRNAs. EE housing blocks pro-inflammatory cytokine gene induction and promotes arginase 1 mRNA expression in brain-sorted microglia, indicating that EE favors an anti-inflammatory activation state. We show that microglia and brain-macrophages from corticosterone-treated mice adopt differential expression profiles for CCR2, MHC class II and IL-4recα surface markers depending on whether the mice are kept in standard environment or EE. Interestingly, the effects of EE were abolished when cells are isolated from ApN knock-out mouse brains. When injected intra-cerebroventricularly, ApN, whose level is specifically increased in cerebrospinal fluid of depressive mice raised in EE, rescues microglia phenotype, reduces pro-inflammatory cytokine production by microglia and blocks depressive-like behavior in corticosterone-treated mice. Our data suggest that EE-induced ApN increase within the brain regulates microglia and brain macrophages phenotype and activation state, thus reducing neuroinflammation and depressive-like behaviors in mice.

  17. Regulations of enzymes in animals: effects of developmental processes, cancer and radiation. Progress report X, 1 May 1975--30 April 1976

    Energy Technology Data Exchange (ETDEWEB)

    Knox, W.E.

    1976-06-01

    The accumulated analysis of tissues shows statistically significant discriminations between normal and neoplastic tissues of a variety of types. The practical identification of cancer by chemical means is thus possible, in principle. The principle itself is of immediate importance: that cancers share a common chemical pattern that can be sought in diagnostic studies, and targeted for therapeutic manipulations. To pursue these studies, additional varieties of transplantable neoplasms were produced and described. The common composition shared by neoplasms bears many resemblances to that of normal immature tissues, and this has reinforced interest in the programmed gene expressions we have studied during development. The development of physiological functions in parallel with the appearance of enzyme components in differentiating tissues has also been a fertile field. Instances of gene mutation associated with absence of an enzyme and consequently with severe functional impairment have been simpler to analyze. A study of phenylketonuria was begun, this time focussing our knowledge of the regulation of gene expression to produce an experimental model of the diesease. A new mutation in the rat causing infantile ichthyosis was identified and isolated. One sign of the disease is hyperkeratosis, analogous to that seen in preneoplastic lesions. The susceptibility of these animals to carcinogenesis in the skin is being studied. The quantitative analysis of numerous enzymes in various tissues has disclosed a variety of new isozymes which have been studied sufficiently to define them as chemically new species and to survey their possible functional importance. The include the several glutaminases, glutamine synthetase and its associated transferase isozyme (glutamine hydroxylamine transferase), and the new arginase of non-hepatic tissues. car

  18. The effect of chemically modified alginates on macrophage phenotype and biomolecule transport.

    Science.gov (United States)

    Bygd, Hannah C; Bratlie, Kaitlin M

    2016-07-01

    Macrophage (MΦ) reprogramming has received significant attention in applications such as cancer therapeutics and tissue engineering where the host immune response to biomaterials is crucial in determining the success or failure of an implanted device. Polymeric systems can potentially be used to redirect infiltrating M1 MΦs toward a proangiogenic phenotype. This work exploits the concept of MΦ reprogramming in the engineering of materials for improving the longevity of tissue engineering scaffolds. We have investigated the effect of 13 different chemical modifications of alginate on MΦ phenotype. Markers of the M1 response-tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase-and the M2 response-arginase-were measured and used to determine the ability of the materials to alter MΦ phenotype. It was found that some modifications were able to reduce the pro-inflammatory response of M1 MΦs, others appeared to amplify the M2 phenotype, and the results for two materials suggested they were able to reprogram a MΦ population from M1 to M2. These findings were supplemented by studies done to examine the permselectivity of the materials. Diffusion of TNF-α was completely prevented through some of these materials, while up to 84% was found to diffuse through others. The diffusion of insulin through the materials was statistically consistent. These results suggest that the modification of these materials might alter mass transport in beneficial ways. The ability to control polarization of MΦ phenotypes with immunoprotective materials has the potential to augment the success of tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1707-1719, 2016.

  19. Metabolic reprogramming and inflammation act in concert to control vascular remodeling in hypoxic pulmonary hypertension.

    Science.gov (United States)

    Stenmark, Kurt R; Tuder, Rubin M; El Kasmi, Karim C

    2015-11-15

    Pulmonary hypertension (PH) is a complex, multifactorial syndrome that remains poorly understood despite decades of research. PH is characterized by profound pulmonary artery (PA) remodeling that includes significant fibro-proliferative and inflammatory changes of the PA adventitia. In line with the emerging concept that PH shares key features with cancer, recent work centers on the idea that PH results from a multistep process driven by reprogramming of gene-expression patterns that govern changes in cell metabolism, inflammation, and proliferation. Data demonstrate that in addition to PA endothelial cells and smooth muscle cells, adventitial fibroblasts from animals with experimental hypoxic PH and from humans with PH (hereafter, termed PH-Fibs) exhibit proinflammatory activation, increased proliferation, and apoptosis resistance, all in the context of metabolic reprogramming to aerobic glycolysis. PH-Fibs can also recruit, retain, and activate naïve macrophages (Mϕ) toward a proinflammatory/proremodeling phenotype through secretion of chemokines, cytokines, and glycolytic metabolites, among which IL-6 and lactate play key roles. Furthermore, these fibroblast-activated Mϕ (hereafter, termed FAMϕ) exhibit aerobic glycolysis together with high expression of arginase 1, Vegfa, and I1lb, all of which require hypoxia-inducible factor 1α and STAT3 signaling. Strikingly, in situ, the adventitial Mϕ phenotype in the remodeled PA closely resembles the Mϕ phenotype induced by fibroblasts in vitro (FAMϕ), suggesting that FAMϕ crosstalk involving metabolic and inflammatory signals is a critical, pathogenetic component of vascular remodeling. This review discusses metabolic and inflammatory changes in fibroblasts and Mϕ in PH with the goal of raising ideas about new interventions to abrogate remodeling in hypoxic forms of PH.

  20. Acute cysticercosis favours rapid and more severe lesions caused by Leishmania major and Leishmania mexicana infection, a role for alternatively activated macrophages.

    Science.gov (United States)

    Rodríguez-Sosa, Miriam; Rivera-Montoya, Irma; Espinoza, Arlett; Romero-Grijalva, Miriam; López-Flores, Roberto; González, Jorge; Terrazas, Luis I

    2006-08-01

    Parasitic helminths have developed complex mechanisms to modulate host immunity. In the present study we found that previous infection of mice with the cestode Taenia crassiceps favours parasitemia and induces larger cutaneous lesions during both Leishmania major and Leishmania mexicana co-infections. Analysis of cytokine responses into draining lymph nodes indicated that co-infection of T. crassiceps-Leishmania did not inhibit IFN-gamma production in response to Leishmania antigens, but significantly increased IL-4 production. Additionally, anti-Leishmania-specific IgG1 antibodies and total IgE increased in co-infected mice, whereas, IgG2a titers remained similar. Macrophages from Taenia-infected mice displayed increased mRNA transcripts of arginase-1, Ym1, and Mannose Receptor, as well as greater production of urea (all markers for an alternate activation state) compared to macrophages from Leishmania-infected mice. In contrast, lower mRNA transcripts for IL-12p35, IL-12p40, IL-23p19, and iNOS were detected in macrophages obtained from cestode-infected mice compared to uninfected and Leishmania-infected mice after LPS stimulation. The presence of cestode also generated impaired macrophage anti-leishmanicidal activity in vitro, as evidenced by the inability of these macrophages to prevent Leishmania growth compared to macrophages from uninfected mice. This was observed despite the fact that both groups of cells were exposed to IFN-gamma. Flow cytometry showed high IFN-gammaR expression on Taenia-induced macrophages. Thus, lack of response to IFN-gamma is not associated with the absence of its receptor. Our data suggest that cestode infection may favour Leishmania installation by inducing alternatively activated macrophages rather than inhibiting Th1-type responses.

  1. Immunotherapy: Beyond Anti-PD-1 and Anti-PD-L1 Therapies.

    Science.gov (United States)

    Antonia, Scott J; Vansteenkiste, Johan F; Moon, Edmund

    2016-01-01

    Advanced-stage non-small cell lung cancer (NSCLC) and small cell lung cancer are cancers in which chemotherapy produces a survival benefit, although it is small. We now know that anti-PD-1/PD-L1 has substantial clinical activity in both of these diseases, with an overall response rate (ORR) of 15%-20%. These responses are frequently rapid and durable, increase median overall survival (OS) compared with chemotherapy, and produce long-term survivors. Despite these very significant results, many patients do not benefit from anti-PD-1/PD-L1. This is because of the potential for malignancies to co-opt myriad immunosuppressive mechanisms other than aberrant expression of PD-L1. Conceptually, these can be divided into three categories. First, for some patients there is likely a failure to generate sufficient functional tumor antigen-specific T cells. Second, for others, tumor antigen-specific T cells may be generated but fail to enter into the tumor parenchyma. Finally, there are a large number of immunosuppressive mechanisms that have the potential to be operational within the tumor microenvironment: surface membrane immune checkpoint proteins PD-1, CTLA-4, LAG3, TIM3, BTLA, and adenosine A2AR; soluble factors and metabolic alterations interleukin (IL)-10, transforming growth factor (TGF)-β, adenosine, IDO, and arginase; and inhibitory cells, cancer-associated fibroblasts (CAFs), regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages. In this article, we discuss three strategies to generate more tumor-reactive T cells for patients: anti-CTLA-4, therapeutic tumor vaccination, and adoptive cellular therapy, with T cells redirected to tumor antigens using T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene modification. We also review some of the various strategies in development to thwart tumor microenvironment immunosuppressive mechanisms. Strategies to drive more T cells into tumors remain a significant challenge.

  2. Immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation.

    Science.gov (United States)

    Miró-Mur, Francesc; Pérez-de-Puig, Isabel; Ferrer-Ferrer, Maura; Urra, Xabier; Justicia, Carles; Chamorro, Angel; Planas, Anna M

    2016-03-01

    Acute stroke induces a local inflammatory reaction causing leukocyte infiltration. Circulating monocytes are recruited to the ischemic brain and become tissue macrophages morphologically indistinguishable from reactive microglia. However, monocytes are a heterogeneous population of cells with different functions. Herein, we investigated the infiltration and fate of the monocyte subsets in a mouse model of focal brain ischemia by permanent occlusion of the distal portion of the middle cerebral artery. We separated two main subtypes of CD11b(hi) monocytes according to their expression of the surface markers Ly6C and CD43. Using adoptive transfer of reporter monocytes and monocyte depletion, we identified the pro-inflammatory Ly6C(hi)CD43(lo)CCR2(+) subset as the predominant monocytes recruited to the ischemic tissue. Monocytes were seen in the leptomeninges from where they entered the cortex along the penetrating arterioles. Four days post-ischemia, they had invaded the infarcted core, where they were often located adjacent to blood vessels. At this time, Iba-1(-) and Iba-1(+) cells in the ischemic tissue incorporated BrdU, but BrdU incorporation was rare in the reporter monocytes. The monocyte phenotype progressively changed by down-regulating Ly6C, up-regulating F4/80, expressing low or intermediate levels of Iba-1, and developing macrophage morphology. Moreover, monocytes progressively acquired the expression of typical markers of alternatively activated macrophages, like arginase-1 and YM-1. Collectively, the results show that stroke mobilized immature pro-inflammatory Ly6C(hi)CD43(lo) monocytes that acutely infiltrated the ischemic tissue reaching the core of the lesion. Monocytes differentiated to macrophages with features of alternative activation suggesting possible roles in tissue repair during the sub-acute phase of stroke.

  3. Potential impact of Paracentrotus lividus extract on diabetic rat models induced by high fat diet/streptozotocin

    Directory of Open Access Journals (Sweden)

    Amel M. Soliman

    2016-10-01

    Full Text Available Antioxidant therapy has been thought to be effectual for the prevention and treatment of various diseases including diabetes. Therefore, the present study was designed to investigate the potency of Paracentrotus lividus extract (PLE for alleviating the complications that resulted after induction of the diabetic rat models (T1DM and T2DM using high fat diet (HFD/streptozotocin (STZ. Thirty six male Wistar albino rats were assigned into normal control, T1DM and T2DM untreated, and PLE treated diabetic rat groups. Induction of T1DM was performed by streptozotocin injection (60 mg/kg of dissolved in sodium citrate buffer, 0.1 mol/L, i.p. T2DM induction through 4 weeks of high fat diet (HFD intervention was followed by a single low dosage of STZ (30 mg/kg dissolved in 0.1 mol/L citrate buffer at pH 4.5, i.p. Both diabetic rat models showed a significant increase in serum; levels of fasting glucose, total protein, bilirubin, activities of arginase, transaminases (AST and ALT, alkaline phosphatase (ALP, γ glutamyl transferase (GGT, lipid profile parameters, and liver malondialdehyde (MDA. However, T1DM and T2DM rats have decreased levels of serum insulin, and liver glucose 6 phosphate dehydrogenase (G6PD, glutathione reduced (GSH, nitric oxide (NO, and antioxidant enzymes. Furthermore, the present study showed the hypoglycemic, hypolipidemic, and antioxidant potency of the PLE as confirmed by its ability for ameliorating most of the alterations caused in the studied parameters of diabetic rats. In conclusion, PLE may be useful as therapy against oxidative stress and liver damage in both types of diabetes mellitus and is therefore recommended for further studies.

  4. The Role of Peritoneal Alternatively Activated Macrophages in the Process of Peritoneal Fibrosis Related to Peritoneal Dialysis

    Directory of Open Access Journals (Sweden)

    Jie Wang

    2013-05-01

    Full Text Available It has been confirmed that alternatively activated macrophages (M2 participate in tissue remodeling and fibrosis occurrence, but the effect of M2 on peritoneal fibrosis related to peritoneal dialysis (PD hasn’t been elucidated. This study was therefore conducted to assess the association between M2 and peritoneal fibrosis related to PD. In this study, peritoneal fibrosis was induced by intraperitoneal (i.p. injection of Lactate-4.25% dialysate (100 mL/kg to C57BL/6J mice for 28 days, and liposome-encapsulated clodronate (LC, the specific scavenger of macrophages was used to treat the peritoneal fibrosis mice model by i.p. injection at day 18 and day 21. All animals were sacrificed at day 29. Parietal peritonea were stained with Masson’s trichrome, and the expression of type I collagen (Col-I, fibronectin, mannose receptor (CD206, transforming growth factor beta (TGF-β, chemokine receptor 7 (CCR7, chitinase 3-like 3 (Ym-1 and arginase-1 (Arg-1 was determined by Western blotting, immunofluorescence and quantitative real-time PCR. Our results revealed that peritoneal thickness, Col-I, fibronectin, CD206, TGF-β, Ym-1 and Arg-1 were upregulated in the peritoneal fibrosis mice model, and all of these indexes were downregulated in those treated with LC. Additionally, there was no difference in the level of CCR7 between the model and treatment group. Our study indicated that peritoneal M2 played an important role in the process of peritoneal fibrosis related to PD and might be a potential target for intervention therapy of peritoneal fibrosis.

  5. Increased erythrocytes by-products of arginine catabolism are associated with hyperglycemia and could be involved in the pathogenesis of type 2 diabetes mellitus.

    Science.gov (United States)

    Ramírez-Zamora, Serafín; Méndez-Rodríguez, Miguel L; Olguín-Martínez, Marisela; Sánchez-Sevilla, Lourdes; Quintana-Quintana, Miguel; García-García, Norberto; Hernández-Muñoz, Rolando

    2013-01-01

    Diabetes mellitus (DM) is a worldwide disease characterized by metabolic disturbances, frequently associated with high risk of atherosclerosis and renal and nervous system damage. Here, we assessed whether metabolites reflecting oxidative redox state, arginine and nitric oxide metabolism, are differentially distributed between serum and red blood cells (RBC), and whether significant metabolism of arginine exists in RBC. In 90 patients with type 2 DM without regular treatment for diabetes and 90 healthy controls, paired by age and gender, we measured serum and RBC levels of malondialdehyde (MDA), nitrites, ornithine, citrulline, and urea. In isolated RBC, metabolism of L-[(14)C]-arginine was also determined. In both groups, nitrites were equally distributed in serum and RBC; citrulline predominated in serum, whereas urea, arginine, and ornithine were found mainly in RBC. DM patients showed hyperglycemia and increased blood HbA1C, and increased levels of these metabolites, except for arginine, significantly correlating with blood glucose levels. RBC were observed to be capable of catabolizing arginine to ornithine, citrulline and urea, which was increased in RBC from DM patients, and correlated with an increased affinity for arginine in the activities of putative RBC arginase (Km = 0.23±0.06 vs. 0.50±0.13 mM, in controls) and nitric oxide synthase (Km = 0.28±0.06 vs. 0.43±0.09 mM, in controls). In conclusion, our results suggest that DM alters metabolite distribution between serum and RBC, demonstrating that RBC regulate serum levels of metabolites which affect nitrogen metabolism, not only by transporting them but also by metabolizing amino acids such as arginine. Moreover, we confirmed that urea can be produced also by human RBC besides hepatocytes, being much more evident in RBC from patients with type 2 DM. These events are probably involved in the specific physiopathology of this disease, i.e., endothelial damage and dysfunction.

  6. A Coral-Derived Compound Improves Functional Recovery after Spinal Cord Injury through Its Antiapoptotic and Anti-Inflammatory Effects

    Science.gov (United States)

    Chen, Chun-Hong; Chen, Nan-Fu; Feng, Chien-Wei; Cheng, Shu-Yu; Hung, Han-Chun; Tsui, Kuan-Hao; Hsu, Chi-Hsin; Sung, Ping-Jyun; Chen, Wu-Fu; Wen, Zhi-Hong

    2016-01-01

    Background: Our previous in vitro results demonstrated that 11-dehydrosinulariolide significantly reduced 6-hydroxydopamine-induced cytotoxicity and apoptosis in a human neuroblastoma cell line, SH-SY5Y, and suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 in lipopolysaccharide-stimulated macrophage cells. The neuroprotective and anti-inflammatory effects of 11-dehydrosinulariolide may be suitable for treating spinal cord injury (SCI). Methods: In the present study, Wistar rats were pretreated with 11-dehydrosinulariolide or saline through intrathecal injection after a thoracic spinal cord contusion injury induced using a New York University (NYU) impactor. The apoptotic cells were assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression and localization of proinflammatory, apoptosis-associated and cell survival-related pathway proteins were examined through immunoblotting and immunohistochemistry. Results: 11-Dehydrosinulariolide attenuated SCI-induced cell apoptosis by upregulating the antiapoptotic protein Bcl-2 and cell survival-related pathway proteins p-Akt and p-ERK, 8 h after SCI. Furthermore, the transcription factor p-CREB, which regulates Bcl-2 expression, was upregulated after 11-dehydrosinulariolide treatment. On day 7 after SCI, 11-dehydrosinulariolide exhibited an anti-inflammatory effect, attenuating SCI-induced upregulation of the inflammatory proteins iNOS and tumor necrosis factor-α. 11-Dehydrosinulariolide also induced an increase in the expression of arginase-1 and CD206, markers of M2 microglia, in the injured spinal cord on day 7 after SCI. Thus, the anti-inflammatory effect of 11-dehydrosinulariolide may be related to the promotion of an alternative pathway of microglia activation. Conclusion: The results show that 11-dehydrosinulariolide exerts antiapoptotic effects at 8 h after SCI and anti-inflammatory effects at 7 days after SCI. We consider that this

  7. 恶性分子标记物的检测与甲状腺结节的诊断%Detection of malignant molecular markers and diagnosis of thyroid nodules

    Institute of Scientific and Technical Information of China (English)

    雷海燕; 王坚

    2010-01-01

    研究发现,许多蛋白分子可以作为甲状腺结节的恶性分子标记物.如半乳糖凝集素-3(galectin-3)、端粒酶(telomerase)、Fra-1蛋白、细胞外基质蛋白1(ECM1)、跨膜丝氨酸蛋白酶4(TMPRSS4)等,以及几种新型生物分子标记物如膜整合蛋白1(ITM1)、1号染色体开放阅读框24(Clorf24)、DNA损伤诱导转录因子3(DDIT3)、精氨酸酶Ⅱ(ARG2).现将甲状腺结节恶性分子标记物的研究进展及其对甲状腺结节的鉴别诊断意义作一综述.%Many protein molecules observed by studies may act as molecular markers of malignant thyroid nodules,such as galectin-3, telomerase, Fra-1 protein, extracellular matrix protein 1 (ECMI), trans-membrane serine protease(TMPRSS4) ,and some new biological molecules, such as integral membrane pro-teinl (ITM1), Chromosome 1 open reading frame 24 (Clorf24), DNA darnage-inducible transcript 3 (DDIT3), arginase Ⅱ (ARG2). In this paper, the progression in the researches of the molecular markers in i-dentification of thyroid nodules are reviewed.

  8. Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia.

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    Santosh K Panda

    Full Text Available Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has

  9. Arterial dysfunction but maintained systemic blood pressure in cavin-1-deficient mice.

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    Karl Swärd

    Full Text Available Caveolae are omega-shaped plasma membrane micro-domains that are abundant in cells of the vascular system. Formation of caveolae depends on caveolin-1 and cavin-1 and lack of either protein leads to loss of caveolae. Mice with caveolin-1 deficiency have dysfunctional blood vessels, but whether absence of cavin-1 similarly leads to vascular dysfunction is not known. Here we addressed this hypothesis using small mesenteric arteries from cavin-1-deficient mice. Cavin-1-reporter staining was intense in mesenteric arteries, brain arterioles and elsewhere in the vascular system, with positive staining of both endothelial and smooth muscle cells. Arterial expression of cavin-1, -2 and -3 was reduced in knockout (KO arteries as was expression of caveolin-1, -2 and -3. Caveolae were absent in the endothelial and smooth muscle layers of small mesenteric arteries as determined by electron microscopy. Arginase, a negative regulator of nitric oxide production, was elevated in cavin-1 deficient arteries as was contraction in response to the α1-adrenergic agonist cirazoline. Detailed assessment of vascular dimensions revealed increased media thickness and reduced distensibility, arguing that enhanced contraction was due to increased muscle mass. Contrasting with increased α1-adrenergic contraction, myogenic tone was essentially absent and this appeared to be due in part to increased nitric oxide production. Vasomotion was less frequent in the knock-out vessels. In keeping with the opposing influences on arterial resistance of increased agonist-induced contractility and reduced myogenic tone, arterial blood pressure was unchanged in vivo. We conclude that deficiency of cavin-1 affects the function of small arteries, but that opposing influences on arterial resistance balance each other such that systemic blood pressure in unstressed mice is well maintained.

  10. In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice.

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    Delphine Hanot Mambres

    Full Text Available Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

  11. Adiponectin and plant-derived mammalian adiponectin homolog exert a protective effect in murine colitis

    KAUST Repository

    Arsenescu, Violeta

    2011-04-11

    Background: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn\\'s disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis. Methods: C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells. Results: Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1β), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid. Conclusion: Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease. © 2011 Springer Science+Business Media, LLC.

  12. Splenic CD4+ T Cells in Progressive Visceral Leishmaniasis Show a Mixed Effector-Regulatory Phenotype and Impair Macrophage Effector Function through Inhibitory Receptor Expression

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    Osorio, Elvia Y.; Saldarriaga, Omar A.; Travi, Bruno L.; Kong, Fanping; Spratt, Heidi; Soong, Lynn

    2017-01-01

    Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL. PMID

  13. CTLA-4Ig immunotherapy of obesity-induced insulin resistance by manipulation of macrophage polarization in adipose tissues

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Masakazu, E-mail: masakazu731079@yahoo.co.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoguchi, Toyoshi, E-mail: toyoshi@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Batchuluun, Battsetseg, E-mail: battsetseg.batchuluun@gmail.com [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Sugiyama, Naonobu, E-mail: nao1@intmed1.med.kyushu-u.ac.jp [Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Kobayashi, Kunihisa, E-mail: nihisak@fukuoka-u.ac.jp [Department of Endocrinology and Diabetes Mellitus, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-8502 (Japan); Sonoda, Noriyuki, E-mail: noriyuki@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Takayanagi, Ryoichi, E-mail: takayana@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2013-08-16

    Highlights: •CTLA-4Ig completely alleviates HFD-induced insulin resistance. •CTLA-4Ig reduces epididymal and subcutaneous fat tissue weight and adipocyte size. •CTLA-4Ig alters ATM polarization from inflammatory M1 to anti-inflammatory M2. •CTLA-4Ig may lead to a novel anti-obesity/inflammation/insulin resistance agent. •We identified the mechanism of the novel favorable effects of CTLA-4lg. -- Abstract: It has been established that obesity alters the metabolic and endocrine function of adipose tissue and, together with accumulation of adipose tissue macrophages, contributes to insulin resistance. Although numerous studies have reported that shifting the polarization of macrophages from M1 to M2 can alleviate adipose tissue inflammation, manipulation of macrophage polarization has not been considered as a specific therapy. Here, we determined whether cytotoxic T-lymphocyte-associated antigen-4IgG1 (CTLA-4Ig) can ameliorate insulin resistance by induction of macrophages from proinflammatory M1 to anti-inflammatory M2 polarization in the adipose tissues of high fat diet-induced insulin-resistant mice. CTLA4-Ig treatment prevented insulin resistance by changing gene expression to M2 polarization, which increased the levels of arginase 1. Furthermore, flow cytometric analysis confirmed the alteration of polarization from CD11c (M1)- to CD206 (M2)-positive cells. Concomitantly, CTLA-4Ig treatment resulted in weight reductions of epididymal and subcutaneous adipose tissues, which may be closely related to overexpression of apoptosis inhibitors in macrophages. Moreover, proinflammatory cytokine and chemokine levels decreased significantly. In contrast, CCAAT enhancer binding protein α, peroxisome proliferator-activated receptor γ, and adiponectin expression increased significantly in subcutaneous adipose tissue. This novel mechanism of CTLA-4lg immunotherapy may lead to an ideal anti-obesity/inflammation/insulin resistance agent.

  14. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

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    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  15. Catabolism and safety of supplemental L-arginine in animals.

    Science.gov (United States)

    Wu, Zhenlong; Hou, Yongqing; Hu, Shengdi; Bazer, Fuller W; Meininger, Cynthia J; McNeal, Catherine J; Wu, Guoyao

    2016-07-01

    L-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76-85 and 81-96 % Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg-HCl or the Arg base [315- and 630-mg Arg/(kg BW d) for 91 d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BW d) for at least 91 d. Intravenous administration of Arg-HCl to gestating sheep at 81 and 180 mg Arg/(kg BW d) is safe for at least 82 and 40 d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BW d) in pigs or 3.6-g Arg/(kg BW d) in rats] for 91 d, which are equivalent to 573-mg Arg/(kg BW d) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans.

  16. Neotuberostemonine attenuates bleomycin-induced pulmonary fibrosis by suppressing the recruitment and activation of macrophages.

    Science.gov (United States)

    Xiang, Juan; Cheng, Si; Feng, Tianlong; Wu, Yan; Xie, Weina; Zhang, Mian; Xu, Xianghong; Zhang, Chaofeng

    2016-07-01

    Neotuberostemonine (NTS) is one of the main antitussive alkaloids in the root of Stemona tuberosa Lour. This study aimed to investigate the effects of NTS on bleomycin (BLM)-induced pulmonary fibrosis in mice and the underlying mechanism. After BLM administration, NTS were orally administered to mice at 20 and 40mg/kg per day from days 8 to 21, with nintedanib as a positive control. The effect of NTS on BLM-induced mice was assessed via histopathological examination by HE and Masson's trichrome staining, TGF-β1 level and macrophage recruitment by immunohistochemical staining, expression of profibrotic media and M1/M2 polarization by western blot. RAW 264.7 cells were used to evaluate whether NTS (1, 10, 100μM) directly affected macrophages. The results revealed that NTS treatment significantly ameliorated lung histopathological changes and decreased inflammatory cell counts in the bronchoalveolar lavage fluid. The over-expression of collagen, α-SMA and TGF-β1 was reduced by NTS. Furthermore, NTS markedly lowered the expression of MMP-2 and TIMP-1 while raised the expression of MMP-9. A further analysis showed that NTS was able to decrease the recruitment of macrophages and to inhibit the M2 polarization in mice lung tissues. The experiment in vitro showed that NTS significantly reduced the arginase-1 (marker for M2) expression in a dose-dependent manner but down-regulated the iNOS (marker for M1) expression only at 100μM. In conclusion, our study demonstrated for the first time that NTS has a significant protective effect on BLM-induced pulmonary fibrosis through suppressing the recruitment and M2 polarization of macrophages.

  17. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development.

    Science.gov (United States)

    Park, Jung-Hyun; Jin, Yoon Mi; Hwang, Soojin; Cho, Du-Hyong; Kang, Duk-Hee; Jo, Inho

    2013-08-01

    The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

  18. Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species.

    Science.gov (United States)

    Kusmartsev, Sergei; Nefedova, Yulia; Yoder, Daniel; Gabrilovich, Dmitry I

    2004-01-15

    Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-gamma production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.

  19. Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

    Science.gov (United States)

    Cammack, R.; Shergill, J. K.; Ananda Inalsingh, V.; Hughes, Martin N.

    1998-12-01

    Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS) 2Fe(NO) 2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.

  20. Dexmedetomidine Regulates 6-hydroxydopamine-Induced Microglial Polarization.

    Science.gov (United States)

    Zhang, Pei; Li, Yu; Han, Xuechang; Xing, Qunzhi; Zhao, Lei

    2017-02-28

    Microglia have undergone extensive characterization and have been shown to present distinct phenotypes, such as the M1 or M2 phenotypes, depending on their stimuli. As a highly specific neurotoxin, 6-hydroxydopamine (6-OHDA) can be used to further our understanding of the immune response in Parkinson's disease (PD). Dexmedetomidine (DEX), a centrally selective α2-adrenoceptor agonist, performs very well as an anti-anxiety medication, sedative and analgesic. In the present study, we investigated the effects of DEX on 6-OHDA-induced microglial polarization. Our results indicate that treatment with 6-OHDA promotes microglial polarization toward the M1 state in BV2 microglia cells by increasing the release of interleukin (IL)-6, IL-1β, or tumor necrosis factor-α, which can be prevented by pretreatment with DEX. In addition, we found that 6-OHDA blocked IL-4-mediated microglial M2 polarization by suppressing expression of the microglial M2 markers arginase-1 (Arg-1), resistin-like α (Retnla/Fizz1), and chitinase 3-like 3 (Chi3l3/Ym1), which could be ameliorated by pretreatment with DEX. Notably, the inhibitory effects of 6-OHDA on IL-4-mediated induction of the anti-inflammatory marker genes IL-10, IL-13, and transforming growth factor-β2 could be significantly alleviated by pretreatment with DEX in a dose-dependent manner (P < 0.01). Mechanistically, alternations in the activation of signal transducer and activator of transcription 6 were involved in this process. These findings suggest that administration of DEX has the potential to interrupt the process of microgliosis in PD.

  1. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

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    Cindy Franklin

    Full Text Available Chronic graft-versus-host disease (cGVHD is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

  2. Redox regulation in metabolic programming and inflammation.

    Science.gov (United States)

    Griffiths, Helen R; Gao, Dan; Pararasa, Chathyan

    2017-02-12

    Energy metabolism and redox state are intrinsically linked. In order to mount an adequate immune response, cells must have an adequate and rapidly available energy resource to migrate to the inflammatory site, to generate reactive oxygen species using NADPH as a cofactor and to engulf bacteria or damaged tissue. The first responder cells of the innate immune response, neutrophils, are largely dependent on glycolysis. Neutrophils are relatively short-lived, dying via apoptosis in the process of bacterial killing through production of hypochlorous acid and release of extracellular NETs. Later on, the most prevalent recruited innate immune cells are monocytes. Their role is to complete a damage limitation exercise initiated by neutrophils and then, as re-programmed M2 macrophages, to resolve the inflammatory event. Almost twenty five years ago, it was noted that macrophages lose their glycolytic capacity and become anti-inflammatory after treatment with corticosteroids. In support of this we now understand that, in contrast to early responders, M2 macrophages are predominantly dependent on oxidative phosphorylation for energy. During early inflammation, polarisation towards M1 macrophages is dependent on NOX2 activation which, via protein tyrosine phosphatase oxidation and AKT activation, increases trafficking of glucose transporters to the membrane and consequently increases glucose uptake for glycolysis. In parallel, mitochondrial efficiency is likely to be compromised via nitrosylation of the electron transport chain. Resolution of inflammation is triggered by encounter with apoptotic membranes exposing oxidised phosphatidylserine that interact with the scavenger receptor, CD36. Downstream of CD36, activation of AMPK and PPARγ elicits mitochondrial biogenesis, arginase expression and a switch towards oxidative phosphorylation in the M2 macrophage. Proinflammatory cytokine production by M2 cells decreases, but anti-inflammatory and wound healing growth factor

  3. Microarray analysis of tomato plants exposed to the nonviruliferous or viruliferous whitefly vector harboring Pepper golden mosaic virus.

    Science.gov (United States)

    Musser, Richard O; Hum-Musser, Sue M; Gallucci, Matthew; DesRochers, Brittany; Brown, Judith K

    2014-01-01

    Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) (Begomovirus, Geminiviridae). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated "known" genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect-plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection

  4. Dietary soyasaponin supplementation to pea protein concentrate reveals nutrigenomic interactions underlying enteropathy in Atlantic salmon (Salmo salar

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    Kortner Trond M

    2012-07-01

    Full Text Available Abstract Background Use of plant ingredients in aquaculture feeds is impeded by high contents of antinutritional factors such as saponins, which may cause various pharmacological and biological effects. In this study, transcriptome changes were analyzed using a 21 k oligonucleotide microarray and qPCR in the distal intestine of Atlantic salmon fed diets based on five plant protein sources combined with soybean saponins. Results Diets with corn gluten, sunflower, rapeseed or horsebean produced minor effects while the combination of saponins with pea protein concentrate caused enteritis and major transcriptome changes. Acute inflammation was characterised by up-regulation of cytokines, NFkB and TNFalpha related genes and regulators of T-cell function, while the IFN-axis was suppressed. Induction of lectins, complement, metalloproteinases and the respiratory burst complex parallelled a down-regulation of genes for free radical scavengers and iron binding proteins. Marked down-regulation of xenobiotic metabolism was also observed, possibly increasing vulnerability of the intestinal tissue. A hallmark of metabolic changes was dramatic down-regulation of lipid, bile and steroid metabolism. Impairment of digestion was further suggested by expression changes of nutrient transporters and regulators of water balance (e.g. aquaporin, guanylin. On the other hand, microarray profiling revealed activation of multiple mucosal defence processes. Annexin-1, with important anti-inflammatory and gastroprotective properties, was markedly up-regulated. Furthermore, augmented synthesis of polyamines needed for cellular proliferation (up-regulation of arginase and ornithine decarboxylase and increased mucus production (down-regulation of glycan turnover and goblet cell hyperplasia could participate in mucosal healing and restoration of normal tissue function. Conclusion The current study promoted understanding of salmon intestinal pathology and establishment of a

  5. Gr1(intCD11b+ myeloid-derived suppressor cells in Mycobacterium tuberculosis infection.

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    Andrés Obregón-Henao

    Full Text Available BACKGROUND: Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(intCD11b(+ cells are called myeloid-derived suppressor cells (MDSC and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design. METHODOLOGY/PRINCIPAL FINDINGS: We compared the bacterial burden, lung pathology and Gr1(intCD11b(+ myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+ cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+CD11b(+ cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi and Gr1(int populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi and Gr1(int cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+ T to Gr1(+ cells increased. Our results

  6. Gr1intCD11b+ Myeloid-Derived Suppressor Cells in Mycobacterium tuberculosis Infection

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    Obregón-Henao, Andrés; Henao-Tamayo, Marcela; Orme, Ian M.; Ordway, Diane J.

    2013-01-01

    Background Tuberculosis is one of the world’s leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1intCD11b+ cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design. Methodology/Principal Findings We compared the bacterial burden, lung pathology and Gr1intCD11b+ myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1+ cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1+CD11b+ cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1hi and Gr1int populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1int populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1hi and Gr1int cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4+ T to Gr1+ cells increased. Our results illustrate a yet unrecognized interplay

  7. Exacerbation of diabetic renal alterations in mice lacking vasohibin-1.

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    Norikazu Hinamoto

    Full Text Available Vasohibin-1 (VASH1 is a unique endogenous inhibitor of angiogenesis that is induced in endothelial cells by pro-angiogenic factors. We previously reported renoprotective effect of adenoviral delivery of VASH1 in diabetic nephropathy model, and herein investigated the potential protective role of endogenous VASH1 by using VASH1-deficient mice. Streptozotocin-induced type 1 diabetic VASH1 heterozygous knockout mice (VASH1(+/- or wild-type diabetic mice were sacrificed 16 weeks after inducing diabetes. In the diabetic VASH1(+/- mice, albuminuria were significantly exacerbated compared with the diabetic wild-type littermates, in association with the dysregulated distribution of glomerular slit diaphragm related proteins, nephrin and ZO-1, glomerular basement membrane thickening and reduction of slit diaphragm density. Glomerular monocyte/macrophage infiltration and glomerular nuclear translocation of phosphorylated NF-κB p65 were significantly exacerbated in the diabetic VASH1(+/- mice compared with the diabetic wild-type littermates, accompanied by the augmentation of VEGF-A, M1 macrophage-derived MCP-1 and phosphorylation of IκBα, and the decrease of angiopoietin-1/2 ratio and M2 macrophage-derived Arginase-1. The glomerular CD31(+ endothelial area was also increased in the diabetic VASH1(+/- mice compared with the diabetic-wild type littermates. Furthermore, the renal and glomerular hypertrophy, glomerular accumulation of mesangial matrix and type IV collagen and activation of renal TGF-β1/Smad3 signaling, a key mediator of renal fibrosis, were exacerbated in the diabetic VASH1(+/- mice compared with the diabetic wild-type littermates. In conditionally immortalized mouse podocytes cultured under high glucose condition, transfection of VASH1 small interfering RNA (siRNA resulted in the reduction of nephrin, angiopoietin-1 and ZO-1, and the augmentation of VEGF-A compared with control siRNA. These results suggest that endogenous VASH1 may

  8. Plasma levels of arginine, ornithine, and urea and growth performance of broilers fed supplemental L-arginine during cool temperature exposure.

    Science.gov (United States)

    Ruiz-Feria, C A; Kidd, M T; Wideman, R F

    2001-03-01

    higher plasma levels of urea and ornithine compared with low-CP diets. These results indicate that kidney arginase was readily activated by Arg provided in the water, resulting in an immediate increase in plasma urea and ornithine. Plasma Arg was increased significantly, but no effects were observed in ascites mortality.

  9. Hypertonic saline resuscitation contributes to early accumulation of circulating myeloid-derived suppressor cells in a rat model of hemorrhagic shock

    Institute of Scientific and Technical Information of China (English)

    LU Yuan-qiang; GU Lin-hui; ZHANG Qin; JIANG Jiu-kun; MOU Han-zhou

    2013-01-01

    Background Hemorrhagic shock is usually associated with complicated immune and inflammatory responses,which are sometimes crucial for the prognosis.As regulators of the immune and inflammatory system; proliferation,migration,distribution and activation of myeloid-derived suppressor cells (MDSCs) are intimately linked to the inflammation cascade.Methods In a model of severe hemorrhagic shock,thirty-five rats were randomly divided into control,sham,normal saline resuscitation (NS),hypertonic saline resuscitation (HTS),and hydroxyethyl starch resuscitation (HES),with seven in each group.M DSCs were analyzed by flow cytometric staining of CD11b/c+Gra+ in peripheral blood mononuclear cells (PBMC),spleen cell suspensions,and bone marrow nucleated cells (BMNC).Simultaneously,the expressions of arginase-1 (ARG-1) and inducible nitric oxide synthase (iNOS) mRNA in MDSCs were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).Results In the early stage after hemorrhagic shock,fluid resuscitation and emergency treatment,the MDSCs in the PBMC of NS,HTS and HES groups markedly increased,and MDSCs in BMNC of these groups decreased accordingly,significantly different to the control group.In hemorrhagic shock rats infused with HTS at the early resuscitation stage,MDSCs in PBMC increased about 2 and 4 folds,and MDSCs in BMNC decreased about 1.3 and 1.6 folds,as compared to the sham group respectively,with statistically significant difference.Furthermore,compared to the NS and HES groups,the MDSCs in PBMC of HTS group increased 1.6 and 1.8 folds with statistically significant differences; the MDSCs decrease in BMNC was not significant.However,there was no statistically significant difference in MDSCs of spleen among the five groups.In addition,compared to the control,sham,NS and HES groups,the ARG-1 and iNOS mRNA of MDSCs in PBMC,spleen and BMNC in the HTS group had the highest level of expression,but no statistically significant differences were

  10. Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse

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    Soper Jessica

    2007-07-01

    Full Text Available Abstract Background Preterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition. Methods Gestational d18.0 uteri (n = 4 were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization. Results We identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri. Conclusion To our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and

  11. Local and systemic gene expression responses of Atlantic salmon (Salmo salar L. to infection with the salmon louse (Lepeophtheirus salmonis

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    Nilsen Frank

    2008-10-01

    Full Text Available Abstract Background The salmon louse (SL is an ectoparasitic caligid crustacean infecting salmonid fishes in the marine environment. SL represents one of the major challenges for farming of salmonids, and veterinary intervention is necessary to combat infection. This study addressed gene expression responses of Atlantic salmon infected with SL, which may account for its high susceptibility. Results The effects of SL infection on gene expression in Atlantic salmon were studied throughout the infection period from copepodids at 3 days post infection (dpi to adult lice (33 dpi. Gene expression was analyzed at three developmental stages in damaged and intact skin, spleen, head kidney and liver, using real-time qPCR and a salmonid cDNA microarray (SFA2. Rapid detection of parasites was indicated by the up-regulation of immunoglobulins in the spleen and head kidney and IL-1 receptor type 1, CD4, beta-2-microglobulin, IL-12β, CD8α and arginase 1 in the intact skin of infected fish. Most immune responses decreased at 22 dpi, however, a second activation was observed at 33 dpi. The observed pattern of gene expression in damaged skin suggested the development of inflammation with signs of Th2-like responses. Involvement of T cells in responses to SL was witnessed with up-regulation of CD4, CD8α and programmed death ligand 1. Signs of hyporesponsive immune cells were seen. Cellular stress was prevalent in damaged skin as seen by highly significant up-regulation of heat shock proteins, other chaperones and mitochondrial proteins. Induction of the major components of extracellular matrix, TGF-β and IL-10 was observed only at the adult stage of SL. Taken together with up-regulation of matrix metalloproteinases (MMP, this classifies the wounds afflicted by SL as chronic. Overall, the gene expression changes suggest a combination of chronic stress, impaired healing and immunomodulation. Steady increase of MMP expression in all tissues except liver was a

  12. Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation.

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    van der Merwe, Marie; Abdelsamed, Hossam A; Seth, Aman; Ong, Taren; Vogel, Peter; Pillai, Asha B

    2013-12-01

    We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4-dependent expansion of donor Foxp3(+) naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2(d)) hosts were administered TLI/ATS and BMT from WT or STAT6(-/-) C57BL/6 (H-2(b)) donors. Following STAT6(-/-) BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4-responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b(+) cell subsets (Gr-1(high)CD11c(-), Gr-1(int)CD11c(-), and Gr-1(low)CD11c(+)) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1(low)CD11c(+) cells induced potent H-2K(b+)CD4(+)Foxp3(+) nTreg proliferation in vitro in 72-h MLRs. Gr-1(low)CD11c(+) cells were reduced significantly in STAT6(-/-) and iNKT cell-deficient Jα18(-/-) BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b(+) cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1(low)CD11c(+) cells to Jα18(-/-) BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3(+) nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-β signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1(low)CD11c(+) cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand-dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.

  13. Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

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    Andrade Marcelle RM

    2012-08-01

    Full Text Available Abstract Background Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. Results Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-γ, inducing proinflammatory type-1 macrophages (M1, or IL-10, inducing anti-inflammatory type-2 cells (M2. Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR and arginase-1 (Arg-1. Treatment of macrophages with IFN-γ and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate

  14. Classical and alternative activation and metalloproteinase expression occurs in foam cell macrophages in male and female ApoE null mice in the absence of T- and B-lymphocytes

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    Elaine Mo Hayes

    2014-10-01

    Full Text Available Background: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1 and alternative (M2 macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs. Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now.Methods and Results: We validated the expression of M1 markers (iNOS and COX-2 and M2 markers (arginase-1, Ym-1 and CD206 and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout and immune-compromised ApoE/Rag-1 double knockout mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14 and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE knockout and ApoE/Rag-1 double knockout mice. Conclusions: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque foam cell macrophages.

  15. LPS converts Gr-1(+)CD115(+) myeloid-derived suppressor cells from M2 to M1 via P38 MAPK.

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    Yang, Yi; Zhang, Ruihua; Xia, Fei; Zou, Ting; Huang, Anfei; Xiong, Sidong; Zhang, Jinping

    2013-07-15

    Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immature myeloid cells with strong immunosuppressive function, and play a critical role in the immune evasion of cancer. A subset of MDSCs share many similar characteristics with tumor-associated macrophages (TAMs), but it is largely unclear whether MDSCs also have M1/M2 type polarization in tumor microenvironments. In the present study, we found that Gr-1(+)CD115(+) monocytes in tumor-bearing mice exhibited M2 characteristics with significantly lower expression of iNOS and higher expression of Arginase I. Immunofluorescence staining showed that Gr-1(+)CD115(+) monocytes in tumor sites from LPS-injected mice had a higher expression of iNOS. Similarly, in vitro experiments displayed that LPS-treated Gr-1(+)CD115(+) cells expressed higher levels of iNOS, IL-6, TNF, IL-12, and IL-10 compared with those in non-treated Gr-1(+)CD115(+) monocytes. Extensive study showed that LPS-treated Gr-1(+)CD115(+) monocytes had less ability to convert the CD4(+)CD25(-)cells into CD4(+)CD25(+) Tregs, and also had less suppressive function on CD4(+)CD25(-) conventional T cells. LLC tumors in LPS-injected mice grew significantly slower than those in non-LPS-injected mice. Further experiments suggested that LPS may function through the P38 MAPK signaling pathway to increase the expression of iNOS, and of MyD88 independently. Thus, we can get conclusion that Gr-1(+)CD115(+) monocytes in tumor-bearing mice show M2 type characteristics and LPS can skew this M2 type cells into M1 type through the P38 MAPK pathway and lead to inhibition of the suppressive function of Gr-1(+)CD115(+) monocytes. It suggests that LPS or its analogs may be potential drugs for tumor treatment, inflammation induced by LPS or other components of bacterium or virus may be benefit to the inhibition of tumor cell growth in vivo.

  16. Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.

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    Ljubica Caldovic

    Full Text Available The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS, carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1 catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C. Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app for acetyl coenzyme A increased while the Km(app for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under

  17. Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803

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    Pistorius Elfriede K

    2007-11-01

    Full Text Available Abstract Background So far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L-arginine catabolism with a transcript analysis. Results We have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L-arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L-arginine-degrading pathways may in principle be functional in this cyanobacterium. These are (i an L-arginine decarboxylase pathway, (ii an L-arginine deiminase pathway, and (iii an L-arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 μmol photons m-2 s-1 showed that the transcripts for the first enzyme(s of all three pathways were present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L-arginine decarboxylase. Conclusion The evaluation of 24

  18. Biphasic Effect of Diabetes on Neuronal Nitric Oxide Release in Rat Mesenteric Arteries

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    Sastre, Esther; Caracuel, Laura; Blanco-Rivero, Javier; Callejo, María; Xavier, Fabiano E.; Balfagón, Gloria

    2016-01-01

    Introduction We analysed possible time-dependent changes in nitrergic perivascular innervation function from diabetic rats and mechanisms implicated. Materials and Methods In endothelium-denuded mesenteric arteries from control and four- (4W) and eight-week (8W) streptozotocin-induced diabetic rats the vasoconstriction to EFS (electrical field stimulation) was analysed before and after preincubation with L-NAME. Neuronal NO release was analysed in the absence and presence of L-arginine, tetrahydrobiopterine (BH4) and L-arginine plus BH4. Superoxide anion (O2-), peroxynitrite (ONOO-) and superoxide dismutase (SOD) activity were measured. Expressions of Cu-Zn SOD, nNOS, p-nNOS Ser1417, p-nNOS Ser847, and Arginase (Arg) I and II were analysed. Results EFS response was enhanced at 4W, and to a lesser extent at 8W. L-NAME increased EFS response in control rats and at 8W, but not at 4W. NO release was decreased at 4W and restored at 8W. L-arginine or BH4 increased NO release at 4W, but not 8W. SOD activity and O2- generation were increased at both 4W and 8W. ONOO- decreased at 4W while increased at 8W. Cu-Zn SOD, nNOS and p-NOS Ser1417 expressions remained unmodified at 4W and 8W, whereas p-nNOS Ser847 was increased at 4W. ArgI was overexpressed at 4W, remaining unmodified at 8W. ArgII expression was similar in all groups. Conclusions Our results show a time-dependent effect of diabetes on neuronal NO release. At 4W, diabetes induced increased O2- generation, nNOS uncoupling and overexpression of ArgI and p-nNOS Ser847, resulting in decreased NO release. At 8W, NO release was restored, involving normalisation of ArgI and p-nNOS Ser847 expressions. PMID:27272874

  19. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone attenuates postincisional pain by regulating macrophage polarization

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa-Moriyama, Maiko, E-mail: hase-mai@m3.kufm.kagoshima-u.ac.jp [Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Ohnou, Tetsuya; Godai, Kohei; Kurimoto, Tae; Nakama, Mayo; Kanmura, Yuichi [Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Rosiglitazone attenuated postincisional pain. Black-Right-Pointing-Pointer Rosiglitazone alters macrophage polarization to F4/80{sup +}CD206{sup +} M2 macrophages at the incisional sites. Black-Right-Pointing-Pointer Transplantation of rosiglitazone-treated macrophages produced analgesic effects. -- Abstract: Acute inflammation triggered by macrophage infiltration to injured tissue promotes wound repair and may induce pain hypersensitivity. Peroxisome proliferator-activated receptor {gamma} (PPAR){gamma} signaling is known to regulate heterogeneity of macrophages, which are often referred to as classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages have considerable antimicrobial activity and produce a wide variety of proinflammatory cytokines. In contrast, M2 macrophages are involved in anti-inflammatory and homeostatic functions linked to wound healing and tissue repair. Although it has been suggested that PPAR{gamma} agonists attenuate pain hypersensitivity, the molecular mechanism of macrophage-mediated effects of PPAR{gamma} signaling on pain development has not been explored. In this study, we investigated the link between the phenotype switching of macrophage polarization induced by PPAR{gamma} signaling and the development of acute pain hypersensitivity. Local administration of rosiglitazone significantly ameliorated hypersensitivity to heat and mechanical stimuli, and paw swelling. Consistent with the down-regulation of nuclear factor {kappa}B (NF{kappa}B) phosphorylation by rosiglitazone at the incisional sites, the number of F4/80{sup +}iNOS{sup +} M1 macrophages was decreased whereas numbers of F4/80{sup +}CD206{sup +} M2 macrophages were increased in rosiglitazone-treated incisional sites 24 h after the procedure. In addition, gene induction of anti-inflammatory M2-macrophage-associated markers such as arginase1, FIZZ1 and interleukin (IL)-10 were significantly increased, whereas

  20. L-glutamine is a key parameter in the immunosuppression phenomenon

    Energy Technology Data Exchange (ETDEWEB)

    Hammami, Ines; Chen, Jingkui [Department of Chemical Engineering, Ecole Polytechnique de Montreal, 2500 Chemin de Polytechnique, Montreal, Quebec, Canada H3T 1J4 (Canada); Bronte, Vincenzo [Department of Pathology, Immunology Section, Verona University, P.le L.A. Scuro, 10 - 37134 Verona (Italy); DeCrescenzo, Gregory [Department of Chemical Engineering, Ecole Polytechnique de Montreal, 2500 Chemin de Polytechnique, Montreal, Quebec, Canada H3T 1J4 (Canada); Jolicoeur, Mario, E-mail: mario.jolicoeur@polymtl.ca [Department of Chemical Engineering, Ecole Polytechnique de Montreal, 2500 Chemin de Polytechnique, Montreal, Quebec, Canada H3T 1J4 (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The absence of L-Gln inhibited iNOS activity, but not ARG1 one. Black-Right-Pointing-Pointer MSC-1 cells were able to inhibit Jurkat cell growth, but not their viability. Black-Right-Pointing-Pointer Absence of L-Gln down-regulated central carbon metabolism and L-Arg recycling. Black-Right-Pointing-Pointer Absence of L-Gln deteriorated cell bioenergetic status. Black-Right-Pointing-Pointer L-Gln is crucial for iNOS-mediated immunosuppression activity. -- Abstract: Suppression of tumour-specific T-cell functions by myeloid-derived suppressor cells (MDSCs) is a dominant mechanism of tumour escape. MDSCs express two enzymes, i.e. inducible nitric oxide synthase (iNOS) and arginase (ARG1), which metabolize the semi-essential amino acid L-arginine (L-Arg) whose bioavailability is crucial for T-cell proliferation and functions. Recently, we showed that glutaminolysis supports MDSC maturation process by ensuring the supply of intermediates and energy. In this work, we used an immortalized cell line derived from mouse MDSCs (MSC-1 cell line) to further investigate the role of L-glutamine (L-Gln) in the maintenance of MDSC immunosuppressive activity. Culturing MSC-1 cells in L-Gln-limited medium inhibited iNOS activity, while ARG1 was not affected. MSC-1 cells inhibited Jukat cell growth without any noticeable effect on their viability. The characterization of MSC-1 cell metabolic profile revealed that L-Gln is an important precursor of lactate production via the NADP{sup +}-dependent malic enzyme, which co-produces NADPH. Moreover, the TCA cycle activity was down-regulated in the absence of L-Gln and the cell bioenergetic status was deteriorated accordingly. This strongly suggests that iNOS activity, but not that of ARG1, is related to an enhanced central carbon metabolism and a high bioenergetic status. Taken altogether, our results suggest that the control of glutaminolysis fluxes may represent a valuable target for immunotherapy.

  1. Characteristic expression of major histocompatibility complex and immune privilege genes in human pluripotent stem cells and their derivatives.

    Science.gov (United States)

    Chen, Hsin-Fu; Yu, Chun-Ying; Chen, Mei-Jou; Chou, Shiu-Huey; Chiang, Ming-Shan; Chou, Wen-Hsi; Ko, Bor-Sheng; Huang, Hsiang-Po; Kuo, Hung-Chih; Ho, Hong-Nerng

    2015-01-01

    Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have been regarded as useful sources for cell-based transplantation therapy. However, immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hESC lines (NTU1 and H9), hiPSC lines, and their derivatives (including stem cell-derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune-related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC-I) molecules, β2-microglobulin, and HLA-E in undifferentiated stem cells. The levels were increased after cotreatment with interferon-γ and/or in vitro differentiation. Antigen-presenting cell markers (CD11c, CD80, and CD86) and MHC-II (HLA-DP, -DQ, and -DR) remained low throughout the treatments. Recognition of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hESCs induced a cell number-dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly, activation of lymphocytes by differentiated hiPSCs or H9 cells became blunted at higher cell numbers. Real-time reverse transcriptase PCR (RT-PCR) showed significant differential expression of immune privilege genes (TGF-β2, Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, IL-1RN, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59, and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It was concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidence suggests

  2. Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells.

    Science.gov (United States)

    Bauer, Georg

    2015-12-01

    Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of

  3. Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

    Science.gov (United States)

    Lee, Sung Won; Park, Hyun Jung; Jeon, Sung Ho; Lee, Changjin; Seong, Rho Hyun; Park, Se-Ho; Hong, Seokmann

    2015-01-01

    Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization

  4. A new metabolomic assay to examine inflammation and redox pathways following LPS challenge

    Directory of Open Access Journals (Sweden)

    Suh Jung H

    2012-10-01

    Full Text Available Abstract Background Shifts in intracellular arginine (Arg and sulfur amino acid (SAA redox metabolism modulate macrophage activation, polarization and phenotype. Despite their importance in inflammation and redox regulatory pathways, comprehensive analysis of these metabolic networks was not previously possible with existing analytical methods. Methods The Arg/thiol redox LC-MS/MS metabolomics assay permits simultaneous assessment of amino acids and derivative products generated from Arg and SAA metabolism. Using this assay, LPS-induced changes in macrophage amino acid metabolism were monitored to identify pathway shifts during activation and their linkage to cellular redox regulation. Results Metabolite concentrations most significantly changed after treatment of a macrophage-like cell line (RAW with LPS for 24 hrs were citrulline (Cit (48-fold increase, ornithine (Orn (8.5-fold increase, arginine (Arg (66% decrease, and aspartic acid (Asp (73% decrease. The ratio Cit + Orn/Arg + Asp (CO/AA was more sensitive to LPS stimulation than other amino acid ratios commonly used to measure LPS-dependent inflammation (e.g., SAM/SAH, GSH/GSSG and total media NOx. The CO/AA ratio was also the first ratio to change significantly after LPS treatment (4 hrs. Changes in the overall metabolomic profile over time indicated that metabolic pathways shifted from Arg catabolism to thiol oxidation. Conclusions Simultaneous quantification of Arg and SAA metabolic pathway shifts following LPS challenge of macrophage indicate that, in this system, the Arg-Citrulline/NO cycle and arginase pathways are the amino acid metabolic pathways most sensitive to LPS-challenge. The cellular (Cit + Orn/(Arg + Asp ratio, which summarizes this pathway, was more responsive to lower concentrations of LPS and responded earlier than other metabolic biomarkers of macrophage activation including GSH redox. It is suggested that the CO/AA ratio is a redox- independent early biomarker of

  5. A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes

    Science.gov (United States)

    Sulaiman, Azad A.; Zolnierczyk, Katarzyna; Japa, Ornampai; Owen, Jonathan P.; Maddison, Ben C.; Hodgkinson, Jane E.; Gough, Kevin C.

    2016-01-01

    The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced

  6. Integration of nondegradable polystyrene and degradable gelatin in a core–sheath nanofibrous patch for pelvic reconstruction

    Directory of Open Access Journals (Sweden)

    Ge LP

    2015-04-01

    phenotype stainings. Qualitative analysis of inducible nitric oxide synthase, arginase, interferon-γ, and interleukin-10 gene expressions also indicates that the implanted composite mesh switches to accommodation mode 2 weeks postimplantation. Thus, these novel core–sheath polystyrene/gelatin nanofibrous membranes are promising in pelvic reconstruction.Keywords: core-sheath nanofibrous patch, polystyrene, gelatin, pelvic reconstruction

  7. Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype

    Science.gov (United States)

    Spratt, Heidi; Travi, Bruno L.; Luxon, Bruce A.

    2017-01-01

    Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even

  8. Biphasic Effect of Diabetes on Neuronal Nitric Oxide Release in Rat Mesenteric Arteries.

    Directory of Open Access Journals (Sweden)

    Esther Sastre

    Full Text Available We analysed possible time-dependent changes in nitrergic perivascular innervation function from diabetic rats and mechanisms implicated.In endothelium-denuded mesenteric arteries from control and four- (4W and eight-week (8W streptozotocin-induced diabetic rats the vasoconstriction to EFS (electrical field stimulation was analysed before and after preincubation with L-NAME. Neuronal NO release was analysed in the absence and presence of L-arginine, tetrahydrobiopterine (BH4 and L-arginine plus BH4. Superoxide anion (O2-, peroxynitrite (ONOO- and superoxide dismutase (SOD activity were measured. Expressions of Cu-Zn SOD, nNOS, p-nNOS Ser1417, p-nNOS Ser847, and Arginase (Arg I and II were analysed.EFS response was enhanced at 4W, and to a lesser extent at 8W. L-NAME increased EFS response in control rats and at 8W, but not at 4W. NO release was decreased at 4W and restored at 8W. L-arginine or BH4 increased NO release at 4W, but not 8W. SOD activity and O2- generation were increased at both 4W and 8W. ONOO- decreased at 4W while increased at 8W. Cu-Zn SOD, nNOS and p-NOS Ser1417 expressions remained unmodified at 4W and 8W, whereas p-nNOS Ser847 was increased at 4W. ArgI was overexpressed at 4W, remaining unmodified at 8W. ArgII expression was similar in all groups.Our results show a time-dependent effect of diabetes on neuronal NO release. At 4W, diabetes induced increased O2- generation, nNOS uncoupling and overexpression of ArgI and p-nNOS Ser847, resulting in decreased NO release. At 8W, NO release was restored, involving normalisation of ArgI and p-nNOS Ser847 expressions.

  9. Reconstruction of liver organoid using a bioreactor

    Institute of Scientific and Technical Information of China (English)

    Masaya Saito; Tomokazu Matsuura; Takahiro Masaki; Haruka Maehashi; Keiko Shimizu; Yoshiaki Hataba; Tohru Iwahori; Tetsuro Suzuki; Filip Braet

    2006-01-01

    AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver.METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 107 FLC-5 cells were incubated in the RFB. After 5 d, 2 x 107 A7 cells were added in a similar manner followed by another addition of 107 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB.RESULTS: By transmission electron microscopy, FLC-5,M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (WO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium,and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased.CONCLUSION: Co-culture in the RFB

  10. Effects of feeding a calf starter on molecular adaptations in the ruminal epithelium and liver of Holstein dairy calves.

    Science.gov (United States)

    Laarman, A H; Ruiz-Sanchez, A L; Sugino, T; Guan, L L; Oba, M

    2012-05-01

    The objective of this study was to elucidate the effect of feeding a calf starter on the volatile fatty acid (VFA) profile in the rumen and on expression of genes involved in epithelial intracellular pH regulation, butyrate metabolism, and hepatic urea cycle during the weaning transition. Twenty Holstein bull calves were fed either milk replacer and hay (MR) or milk replacer, hay, and a commercial texturized calf starter (MR+S) in a randomized complete block design. All calves were fed 750 g/d of milk replacer as the basal diet. Calves on the MR+S treatment were also fed starter ad libitum, and the energy intake of calves within blocks was maintained by supplementing the MR group with extra milk replacer that was equivalent to the energy intake from calf starter. Calves were killed 3 d after they consumed 680 g/d of calf starter for 3 consecutive days. Calves fed MR+S had higher VFA concentrations in the rumen (99.1±8.1 vs. 64.6±8.6 mM) and a higher molar proportion of butyrate (15.6±1.7 vs. 7.9±1.9%) than calves fed MR. Relative abundance of mRNA for monocarboxylate transporter isoform 1 was higher (1.45 vs. 0.53), and that of Na(+)/H(+) exchanger isoform 3 (0.37 vs. 0.82) and 3-hydroxy-3-methylglutaryl synthase isoform 1 (0.40 vs. 0.94) lower for the MR+S treatment compared with the MR treatment. In the liver, relative mRNA abundances of argininosuccinate synthetase isoform 1 (2.67 vs. 1.56), argininosuccinate lyase (1.44 vs. 0.99), and arginase isoform 1 (3.21 vs. 1.74) were greater for MR+S than for MR calves. Calf starter consumption appeared to increase fermentation in the rumen and affected expression of genes involved in cholesterol synthesis and intracellular pH regulation in ruminal epithelium, and those involved in urea cycle in the liver.

  11. L-Proline nutrition and catabolism in Staphylococcus saprophyticus.

    Science.gov (United States)

    Deutch, Charles E

    2011-05-01

    Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require L-proline but not L-arginine for growth in a defined culture medium. All three strains could utilize L-ornithine as a proline source and contained L-ornithine aminotransferase and Δ(1)-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use L-arginine as a proline source and had L-arginase activity. The proline requirement also could be met by L-prolinamide, L-proline methyl ester, and the dipeptides L-alanyl-L-proline and L-leucyl-L-proline. The bacteria exhibited L-proline degradative activity as measured by the formation of Δ(1)-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of L-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller-Hinton broth. A membrane fraction from this strain had L-proline dehydrogenase activity as detected both by reaction of Δ(1)-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min(-1) mg(-1)) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min(-1) mg(-1)). A soluble fraction from this strain had Δ(1)-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min(-1) mg(-1)) as determined by the NAD(+)-dependent oxidation of DL-Δ(1)-pyrroline-5-carboxylate. Addition of L-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with L: -ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-DL-proline, DL-thiazolidine-2-carboxylate, and L-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.

  12. A preliminary study on macrophage plasticity%小鼠巨噬细胞功能极化可塑性的初步探讨

    Institute of Scientific and Technical Information of China (English)

    杨琴; 张志仁; 姜曼; 吴玉章

    2013-01-01

    目的 通过鉴定小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)的功能表型,从而研究巨噬细胞功能极化的可塑性及其内在机制.方法 采用流式细胞术检测体外诱导分化的小鼠骨髓来源巨噬细胞纯度;采用荧光定量PCR技术检测由RAW264.7极化的M1、M2巨噬细胞中M1、M2特定标记基因的表达来鉴定RAW264.7的功能状态;小鼠BMDM极化为M1、M2巨噬细胞时,荧光定量PCR技术检测M1、M2中特定标记基因TNF-α、IL-6、Arginase、Fizz-1 mRNA水平的表达,组蛋白去乙酰化酶抑制剂Trichostatin A(TSA)、DNA去甲基化酶抑制剂5-氮杂-2-脱氧胞(Aza)刺激M1、M2巨噬细胞后,检测TNF-α、IL-6、Arginase、Fizz-1 mRNA水平表达的变化.结果 RAW264.7细胞经LPS刺激8h极化为M1巨噬细胞;RAW264.7细胞经IL-4体外刺激24 h极化为M2巨噬细胞.小鼠BMDM在LPS、IL-4刺激下,分别极化为M1、M2巨噬细胞,改变体外刺激条件,M1巨噬细胞可重分化为M2样巨噬细胞,M2巨噬细胞可重分化为M1样巨噬细胞,M1样巨噬细胞和M2巨噬细胞是介于M1、M2中间状态的2种巨噬细胞;药物TSA、Aza的加入使M1、M2中特定标记基因TNF-α、IL-6、Arginase、Fizz-1 mRNA水平表达增加.结论 巨噬细胞的功能极化具有可塑性,巨噬细胞的极化可能与染色质状态的改变有关.%We aimed to study plasticity of functional differentiation of macrophage and its intrinsic mechanism by identifying functional genotype (M1, M2) of mouse bone marrow-derived macrophages (BMDM). Purity of mouse BMDM induced differently in vitro was detected by flow cytometry; when RAW264.7 polarized to M1 and M2 macrophages, fluorescent quantitation PCR was used to identify the functional status of RAW264.7 by detecting expressions of marked genes in M1 and M2; fluorescent quantitation PCR was used to detect the mRNA expression levels of TNF-α, IL-6, Arginase and Fizz-1 when mouse BMDM polarized to M1 and M2

  13. [The modulation of low-level laser on polarization of mouse bone marrow-derived macrophages].

    Science.gov (United States)

    Dai, Chen; Song, Jiwei; Liang, Zhuowen; Zhang, Qian; Zhang, Kun; Wang, Zhe; Hu, Xueyu

    2016-08-01

    Objective To investigate the influence of 810 nm low-level laser of different energy on the polarization of macrophages. Methods The macrophages were isolated from the bone borrow of BALB/c mice and cultured in macrophage colony stimulating factor (M-CSF) conditioned cultural medium. The expression of F4/80 was examined by flow cytometry for identification. After lipopolysaccharide-γ interferon (LPS-IFN-γ) induced polarization status in the macrophages, the mRNA expressions of inducible nitric oxide synthase (iNOS), arginase 1 (Arg1) and CD86 were detected by reverse transcription PCR, and the protein expressions of iNOS and Arg1 were tested by Western blotting. Thereafter, the M1 macrophages were exposed to 810 nm low-level laser of (1, 2, 3, 4) J/cm(2), and then the cell viability was evaluated by MTT assay; the expressions of iNOS and Arg1 were observed by immunofluorescent cytochemical staining; the mRNA and protein levels of iNOS and Arg1 were studied by reverse transcription PCR and Western blotting. Results Flow cytometry showed that the percentage of F4/80 positive cells cultured with M-CSF conditioned medium was 99.9%. The mRNA and protein levels of iNOS and CD86 in macrophages were both significantly raised after induction by LPS-IFN-γ. Compared with the control cells, the viability of M1 cells significantly decreased when the energy of the low-level laser exposure was 4 J/cm(2), while the viability remained unchanged when the energy was 1, 2 or 3 J/cm(2). Immunocytochemistry revealed that the percentage of Arg1 positive cells that represent M2 macrophages was not significantly different from the control group when the irradiation dose was 1 or 2 J/cm(2), however, the Arg1 positive cells significantly increased and the iNOS positive cells that represent M1 macrophages significantly decreased when the irradiation dose was 3 or 4 J/cm(2). When the irradiation dose was 1 or 2 J/cm(2), the mRNA and protein levels of iNOS and Arg1 remained unchanged

  14. Supplementation with Phycocyanobilin, Citrulline, Taurine, and Supranutritional Doses of Folic Acid and Biotin—Potential for Preventing or Slowing the Progression of Diabetic Complications

    Directory of Open Access Journals (Sweden)

    Mark F. McCarty

    2017-03-01

    Full Text Available Oxidative stress, the resulting uncoupling of endothelial nitric oxide synthase (eNOS, and loss of nitric oxide (NO bioactivity, are key mediators of the vascular and microvascular complications of diabetes. Much of this oxidative stress arises from up-regulated nicotinamide adenine dinucleotide phosphate (NADPH oxidase activity. Phycocyanobilin (PhyCB, the light-harvesting chromophore in edible cyanobacteria such as spirulina, is a biliverdin derivative that shares the ability of free bilirubin to inhibit certain isoforms of NADPH oxidase. Epidemiological studies reveal that diabetics with relatively elevated serum bilirubin are less likely to develop coronary disease or microvascular complications; this may reflect the ability of bilirubin to ward off these complications via inhibition of NADPH oxidase. Oral PhyCB may likewise have potential in this regard, and has been shown to protect diabetic mice from glomerulosclerosis. With respect to oxidant-mediated uncoupling of eNOS, high-dose folate can help to reverse this by modulating the oxidation status of the eNOS cofactor tetrahydrobiopterin (BH4. Oxidation of BH4 yields dihydrobiopterin (BH2, which competes with BH4 for binding to eNOS and promotes its uncoupling. The reduced intracellular metabolites of folate have versatile oxidant-scavenging activity that can prevent oxidation of BH4; concurrently, these metabolites promote induction of dihydrofolate reductase, which functions to reconvert BH2 to BH4, and hence alleviate the uncoupling of eNOS. The arginine metabolite asymmetric dimethylarginine (ADMA, typically elevated in diabetics, also uncouples eNOS by competitively inhibiting binding of arginine to eNOS; this effect is exacerbated by the increased expression of arginase that accompanies diabetes. These effects can be countered via supplementation with citrulline, which efficiently enhances tissue levels of arginine. With respect to the loss of NO bioactivity that contributes to

  15. Arginine metabolism in uricotelic species = Metabolismo da arginina em espécies uricotélicas

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    Jovanir Inês Müller Fernandes

    2010-10-01

    Full Text Available Due to the lack of a complete urea cycle, uricotelic species, such as broilers, are not able to synthesize de novo arginine (Arg, thus depending exclusively on dietary Arg. High levels of dietary lysine (Lys increase the demand for Arg because of the antagonistic relationship between these amino acids. The Arg-Lys antagonism promotes an expressive increase in the renal Arg activity and consequently induces the degradation of Arg and the decrease in theactivity of glycine amidinotransferase, an enzyme that uses Arg in the synthesis of muscle creatin. Arg is considered an important modulator of immunological and physiological processes. The degradation of Arg produces ornithine, a precursor of polyamines that are key to cell division, DNA synthesis, and cell cycle regulation. Arg participates in the synthesis of nitric oxide (NO, a highly reactive free radical in cells and membranes and participates in several cell processes, including in neurotransmission and immune response. Arg is also considered a potent secretagogue of insulin, growth hormone, and IGF-I in the blood stream. Exclusively vegetariandiets may not provide an adequate supply of Arg, which is required for maximum production and for the immune system of current broiler lineages.Devido à falta de um completo ciclo da uréia, espécies uricotélicas como os frangos de corte são incapazes de sintetizar arginina (Arg de novo, por isso dependem exclusivamente da Arg dietética. Níveis elevados de lisina (Lys dietética aumentam a exigência de Arg devido à relação antagônica entre estes aminoácidos. O antagonismo entre Arg e Lys promove expressiva elevação da atividade da arginase renal e conseqüentemente induz à degradação da Arg e leva à diminuição da atividade da glicina amidinotransferase, enzima que utiliza Arg na síntese de creatina muscular. Arg é considerada importante modulador da imunidade e de processos fisiológicos. A degradação de Arg gera ornitina

  16. 微小RNA-155在小鼠骨髓来源M1和M2巨噬细胞中表达的差异%Diverse expression patterns of microRNA-155 between bone marrow-derived M1 and M2 macrophages

    Institute of Scientific and Technical Information of China (English)

    梁艳冰; 王中华; 唐皓; 马中富

    2012-01-01

    BACKGROUND: Previous studies of microRNA-155 expression and function in macrophage predominantly focus on total mononuclear macrophage. OBJCTIVE: To evaluate the expression difference of microRNA-155 in M1 and M2 macrophages. METHODS: Bone marrow-derived macrophages were induced to differentiate t owards M1 and M2 macrophages using interferon-γ(100 U/mL) + lipopolysaccharide (5 ng/mL) and interleukin-4 (10 ng/mL), respectively. RESULTS AND CONCLUSION: The differentiation proportion of M1 and M2 macrophages detected by flow cytometry was 91% and 95%, respectively. Reverse transcription PCR method showed that inducible nitric oxide synthase (iNOS) mRNA was highly expressed in M1 macrophages, but it was hardly expressed in M2 macrophages. Arginase-1 and found in inflammatory zone 1 mRNA expression was highly expressed in M2 macrophages, but the expression was low in M1 macrophages. Tumor necrosis factor α mRNA expression was significantly higher in M1 macrophages than in M2 macrophages, on the contrary, interleukin 10 mRNA expression was significantly higher in M2 macrophages than in M1 macrophages (P < 0.05). Real-time fluorescence quantitative PCR showed that microRNA-155 expression in M1 macrophages was significantly higher compared with that in M2 macrophages (P < 0.05). These findings suggest that MiRNA-155 can serve as a useful maker for differential diagnosis of M1 and M2 macrophages.%背景:目前对微小RNA-155 在巨噬细胞中的表达和功能的研究还集中在总体单核巨噬细胞水平.目的:了解小鼠骨髓来源M1 和M2 巨噬细胞中微小RNA-15 表达的差异.方法:用100 U/mL 干扰素γ和5 μg/L 脂多糖诱导骨髓细胞来源的巨噬细胞向M1 分化,用10 μg/L 白细胞介素4 诱导出M2 巨噬细胞.结果与结论:流式细胞检测显示实验诱导的M1 和M2 巨噬细胞纯度分别达91%和95%.RT-PCR 检测显示诱导型一氧化氮合酶mRNA 在M1 巨噬细胞中高表达,在M2 中则基本不表达;而Ⅰ型精氨

  17. Characterization of iNOS+ Neutrophil-like ring cell in tumor-bearing mice

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    Virtuoso Lauren P

    2012-07-01

    Full Text Available Abstract Background Myeloid-derived Suppressor Cells (MDSC have been identified as tumor-induced immature myeloid cells (IMC with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. Methods Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. Results The results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM, morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid

  18. E ffect of deacetylase inhibitor VPA on polarization of macrophages%去乙酰化酶抑制剂VPA对巨噬细胞极化过程的影响

    Institute of Scientific and Technical Information of China (English)

    张颖; 白力; 张文兰; 尹学红; 张伟; 庞春艳; 王永福

    2016-01-01

    inhibitors whether have the effect on the activity of the macrophage polarization by altered histone modification of macrophages , in order to provide a new perspective for the treatment of autoimmune diseases .Methods:Using lipopolysaccharide ( LPS) and interferon-γ( IFN-γ) to stimulate J774.1 cells for 24 h,and interleukin-4 ( IL-4 ) to stimulate J774.1 cells for 24 h.And 2 mmol/L valproic acid ( VPA ) was added in the induction process.Collecting J774.1 cells,fluorescent quantitation PCR assay and ELISA assay was used for the detection of specific markers of gene expression in macrophage polarization , flow cytometry and immunofluorescence assay for the detection of histone modifications.Results:J774.1 cells were polarized into M1 macrophages which were stimulated by LPS and IFN-γfor 24 h;and also J774.1 cells were polarized into M2 macrophages which were stimulated by IL-4 for 24 h.The degree of acetylation of H 3K9 for M1 phenotype was increased after VPA treatment , the expression of interleukin-6 (IL-6 ) , inducible nitric oxide synthase ( iNOS ) , and chemotactic factor(CCL-2) was decreased,and the expression of CD86 was increased.The degree of acetylation of H3K9 for M1 phenotype was also increased after VPA treatment ,and also the expression of Arginase,Fizz-1,mannose receptor(CD 206) and Ym1 were increased.Conclusion:The polarization state of the macrophages and histone modification had a certain relevance .VPA could induce the transformation of M1 phenotype to M2 phenotype in the induction system of the M1 macrophages,however,the expression of specific genes in M1 phenotype was inhibited in the induction system of the M 2 macrophages.

  19. 青藤碱对LPS、IL-4诱导的小鼠RAW264.7巨噬细胞极化的影响%Effect of sinomenine on mouse RAW264.7 macrophage cells line polarization induced by LPS or IL-4

    Institute of Scientific and Technical Information of China (English)

    罗进芳; 朱瑞丽; 易浪; 董燕; 王培训

    2015-01-01

    Objective:To investigate sinomenine (Sinomenine,SIN) effect on RAW264.7 cells polarization to M1 or M2 phenotype induced by lipopolysaccharide (LPS) or interleukin-4 (IL-4) .Methods:RAW264.7 cells were induced to polarize to M1 by LPS ,and to M2 by IL-4.Sinomenine effects on LPS or IL-4 induced macrophages:TNF-αand IL-10 secretion induced by different condition were detected by Enzyme linked immunosorbent assay (ELISA);The expression level of mRNA of Arginase1(Arg-1),Nitric oxide synthase(iNOS),suppressor of cytokine signaling protein-2(SOCS2) and suppressor of cytokine signaling protein-3(SOCS3) of M1/M2 phenotypes were detected by real time PCR respectively.Results:Sinomenine inhibited the increase of TNF-αsecretion,iNOS and SOCS3 mRNA expression level induced by LPS.Sinomenine inhibited the increase of IL-10 secretion and Arg-1 mRNA expression level induced by IL-4,but SOCS2 mRNA expression level was not affected by Sinomenine.Conclusion: Sinomenine can inhibite the macrophage polarization to M1 and M2 induced by LPS and IL-4.Sinomenine plays a regulatory role on imbalance of M1/M2,and is conducive to maintain the dynamic balance.%目的:探讨青藤碱(Sinomenine,SIN)对脂多糖(LPS)以及白细胞介素4(IL-4)诱导的RAW264.7细胞向M1、M2型极化的影响。方法:以LPS刺激RAW264.7细胞诱导M1型极化,IL-4刺激RAW264.7细胞诱导M2型极化;青藤碱作用于LPS或IL-4诱导的巨噬细胞后:用酶联免疫法( ELISA)检测不同诱导状态下RAW264.7细胞TNF-α和IL-10的分泌量;荧光定量PCR检测与巨噬细胞极化相关的精氨酸酶-1(Arg-1)、一氧化氮合酶(iNOS)、细胞因子信号转导抑制蛋白-2(SOCS2)和细胞因子信号转导抑制蛋白-3(SOCS3)的mRNA表达水平。结果:青藤碱能抑制LPS诱导下细胞TNF-α的分泌量,抑制细胞iNOS和SOCS3的mRNA表达水平的升高。青藤碱能抑制IL-4诱导下细胞IL-10

  20. Effect of Berberine on Mice RAW264.7 Macrophages Polarization%小檗碱对小鼠RAW264.7巨噬细胞极化的影响

    Institute of Scientific and Technical Information of China (English)

    朱瑞丽; 吴阳阳; 罗进芳; 易浪; 董燕

    2014-01-01

    Objective To investigate the effect of berberine on the polarization of mice RAW264.7 macrophages induced separately by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Methods Mice RAW 264.7 macrophages cultured in vitro were divided into model group, medication group, and blank control group. Both model group and medication group were given either LPS (in final dose of 100 ng/mL) or IL-4 (in final dose of 10 ng/mL). Additionally, the medication group was treated with berberine in final dose of 20 μmol/L. The blank control group was given the same volume of phosphate buffered saline ( PBS). Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of arginase-1 (Arg-1), inducible nitric oxide synthase ( iNOS) , suppressor of cytokine signaling2 ( SOCS2) and SOCS3. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of tumor necrosis factor alpha (TNF-α) and IL-10. Results The content of TNF-αand the mRNA expression levels of iNOS and SOCS3 in macrophages induced by LPS were increased, and then were down-regulated by berberine (P0.05). Conclusion Berberine has an effect on inhibiting the M1 and M2 polarization of macrophages in vitro, suggesting that berberine may play a regulatory role in the dynamic balance of M1/M2.%目的观察小檗碱对脂多糖(LPS)、白细胞介素-4(IL-4)诱导的小鼠RAW264.7细胞极化的影响。方法体外培养小鼠巨噬细胞RAW264.7,模型组和给药组分别加入LPS (终浓度100 ng/mL)或IL-4(终浓度10 ng/mL),给药组用终浓度20μmol/L的小檗碱分别进行干预,同时空白对照组给予等体积磷酸盐缓冲液(PBS)。采用荧光定量聚合酶链反应检测精氨酸酶-1( Arg-1)、一氧化氮合酶( iNOS)、细胞因子信号传导抑制蛋白2( SOCS2)、细胞因子信号传导抑制蛋白3(SOCS3)的mRNA表达水平,采用酶联免疫吸附(ELISA)法检测

  1. 腐胺和脯氨酸对哺乳期仔猪空肠绒毛-隐窝轴上皮细胞的多胺代谢及Wnt信号通路的影响%Impact of Putrescine and Proline on Suckling Piglet Jejunum Villus-crypt Axis Epithelial Polyamine Metabolism and Wnt Signal Pathway

    Institute of Scientific and Technical Information of China (English)

    王小城; 熊霞; 杨焕胜; 高巍; 龚敏; 印遇龙

    2015-01-01

    [Objective]The aim of this paper is to study the effects of putrescine and proline on polyamines (putrescine, spermidine and spermine) metabolites, relative mRNA expression levels of Wnt signaling molecules along the villus-crypt axis in the jejunum of sucking piglet.[Method]Eighteen 0-day old newborn crossbred (Landrace × Yorkshire × Duroc) piglets were randomly divided into three groups:control group, putrescine group and proline group. The piglets in control group were administered an equal volume of saline, and that in other two groups received putrescine (5 mg·kg-1 body weight ) and proline (25 mg·kg-1 body weight ), respectively. Piglets were weaned at 14-day old and slaughtered on 3 days postweaning. The jejuna epithelial cells along the crypt-villus axis were isolated by Krebs Henseleit (KH) solution and yield 3“cell fractions”(Fraction 1, 2 and 3). The concentration of polyamine in F1, F2 and F3 was determined by high performance liquid chromatography, and relative mRNA expression of related genes of polyamine metabolic and wnt signaling pathways were measured by real time PCR. [Result] The concentration of putrescine, spermine and spermidine in F1 were significantly increased (P0.05). The concentrations of spermine and spermidine in F2 of proline or putrescine groups were significantly higher than that of control group (P0.05). The expression of ornithine decarboxylase (ODC) gene in F1 of putrescine group was significantly higher than that of proline group and control group (P<0.05). The expression of arginase in F2 of proline group was significantly higher than that of putrescine group and control group (P<0.05). The expression of SFRP3 in F2 of proline group was significantly higher than that of putrescine group (P<0.05);the expression of sFRP4 in F3 of putrescine group is significantly higher than that of control group and proline group (P<0.05). [Conclusion] Exogenous putrescine and proline increased the concentration of polyamine in

  2. Nuevas terapias para el cáncer basadas en el sistema immunitario

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    Pedro Romero

    2011-06-01

    , certain tumor antigens are selected as targets in the design of anticancer vaccine candidates. Until now, hundreds of phase I and II clinical trials have been conducted to test the safety and tolerance of new therapeutic vaccines. Two promising vaccines (MAGE-A3 and MUC-1 are being currently evaluated in terms of their clinical efficiency in treating lung cancer and metastatic melanoma, in randomized, double blind, placebo controlled phase III clinical trials. Third, this information has also inspired the optimization of novel cellular therapies consisting of ex vivo expansion of tumor-infiltrating T lymphocytes for a subsequent reinfusion of massive numbers (billions of these cells. It is also possible to «reprogram» autologous T lymphocytes via transduction with a retroviral vector that carries TCRs specific for defined tumor antigens. At the forefront of basic research, interest centers on understanding the multiple mechanisms operating inside the tumors and which impede an efficient anti-tumoral immune response. New targets have been identified for future therapies that should neutralize these immune-suppressing circuits and, thus, enhance the anti-tumoral response. Among these, there are inhibiting receptors such as CTLA-4, PD-1, Tim-3, BTLA, CD4 regulatory T lymphocytes, or myeloid derived suppressor cells. Certain enzymes are expressed at excessively high levels in the tumor microenvironment like Cox-2, IDO, INOS, and arginase. Their activity suppresses (for different reasons the anti-tumoral immune response. Their inhibition in experimental tumor models has revealed a clear therapeutic potential. Finally, components of tumor stroma such as activated fibroblasts or blood neovessels are also valuable therapeutic targets. In conclusion, cancer immunotherapy is an area undergoing remarkable developments. Recently, new therapies have come about based on harnessing the immune system and it should be expected that in the future the number and efficiency of the new anti

  3. Regulating effect of Notch signaling on macrophage polarization and function in laser-induced CNV%Notch信号对激光诱导的小鼠CNV生成过程中巨噬细胞极化表型及功能的调控

    Institute of Scientific and Technical Information of China (English)

    李娜; 窦国睿; 张萍; 赵俊龙; 晏贤春; 燕洁静; 李曼红; 韩骅; 王雨生

    2015-01-01

    .138,5.272,both at P=0.000).Mφ distributed in both the peripheral and central of CNV in the 3 day group and accumulated in the central of CNV in 7 day group.qRT-PCR assay showed that the relative expressions of arginase 1 (Arg1) mRNA,mannose receptor (MR) mRNA and interleukin 6 (IL-6) mRNA in the CNV were significantly upregulated in post-photocoagulative 3 days and downregulated in 7 days (all at P<0.05).However,no significant shift was found in the expression of iNOS mRNA at the same duration.Immunofluorescence results displayed that there were more F4/80+Arg1 + cells (M2-liked Mφ) than F4/80+iNOS+ cells (M1-liked Mφ) in 3 days and 7 days (t =7.348,4.568,both at P<0.01),and the mount of F4/80+Arg1 + cells was significantly reduced in 7 days compared with in 3 days (t =5.562,P < 0.001).The choroidal flat mounts,qRT-PCR and immunofluorescence results demonstrated that VEGF had a visible expression in CNV in 3 days after photocoagulation,and it was dramatically upregulated in 7 days (t =5.776,P<0.01),but the secreted VEGF level by Mφ was higher in the third day than that in the seventh day (t =5.143,P<0.001).Similarly,the secretion level of TNF-α by Mφ was reclined in the seventh day compared with the third day (t =4.519,P<0.01).In addition,qRT-PCR and immunofluorescence results indicated that the expression of downstream molecule of Notch signaling in Mφ was not found in 3 days,but these molecules were expressed and activated in 7 days after photocoagulation (P<0.05).In vitro,the BMDMs converted to M2 after blockage of Notch signaling,and the decrease of M1 markers and increase of M2 markers were seen (all at P<0.05).Conclusions Mφ polarization phenotypes alter upon the development of CNV.The activation of Notch signaling pathway participates in the modulation of Mφ polarization and functional diversity.%背景 巨噬细胞在脉络膜新生血管(CNV)中的作用尚存在争议,与其在不同微环境中的功能异质性有关,Notch信号通路参与

  4. 负压封闭引流联合含氧液冲洗对下肢慢性静脉性溃疡患者创面的影响%Effects of vacuum sealing drainage combined with irrigation of oxygen loaded fluid on wounds of patients with chronic venous leg ulcers

    Institute of Scientific and Technical Information of China (English)

    温皇鼎; 李志清; 张美光; 王甲汉; 王桂芳; 吴起; 童森

    2015-01-01

    Objective To evaluate the therapeutic effects of VSD combined with irrigation of oxygen loaded fluid on the growth of granulation tissue and macrophage polarization in chronic venous leg ulcers.Methods Thiry-four patients with chronic venous leg ulcers hospitalized in our department from December 2010 to July 2014 were divided into VSD group (A,n =11),VSD+irrigation group (B,n =11),and VSD + oxygen loaded fluid irrigation group (C,n =12) according to the random number table.After admission,debridement was performed,and granulation tissue in the center of the wound was harvested during the operation.After debridement,the patients in group A were treated with VSD only (negative pressure from -30 to-25 kPa,the same below) ; the patients in group B were treated with VSD combining irrigation of normal saline; the patients in group C were treated with VSD combining normal saline loaded with oxygen irrigation (flow of 1 L/min).On post treatment day (PTD) 7,the VSD devices were removed.Gross observation was conducted before debridement and on PTD 7.Ou PTD 7,the granulation tissue in the center of the wound was harvested for histopathological observation with HE staining and Masson staining,following calculation of granulation tissue coverage rate.After debridement but before the negative pressure therapy (hereinafter referred to as before treatment) and on PTD 7,partial pressure of oxygen of the skin around the wound was measured by transcutaneous tissue oxygen tension survey meter.On PTD 7,expression of vascular endothelial growth factor (VEGF) was determined with immunohistochemistry.Before treatment and on PTD 7,cells with double positive expressions of induced nitric oxide synthase plus CD68 (type Ⅰ macrophage) and arginase 1 plus CD68 (type Ⅱ macrophage) were observed with immunofluorescence staining and quantified.Data were processed with Fisher's exact test,one-way analysis of variance,covariance analysis,paired t test,and LSD test.Results (1) The gross

  5. 大鼠全层皮肤缺损创面愈合过程中巨噬细胞浸润及表型研究%Infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat

    Institute of Scientific and Technical Information of China (English)

    牛轶雯; 缪明远; 曹晓赞; 宋菲; 嵇晓芸; 董叫云; 陆树良

    2014-01-01

    -thickness skin defects (11 mm diameter) were created on both sides of dorsal spine of rats in injury group with surgical scissors and homemade trephine.After injury,wound area was measured immediately.The wounds were disinfected with iodophor every day.Rats in control group received anesthesia and hair removal only.On post injury day (PID) 1,3,7,and 13,respectively,6 rats of injury group were sacrificed after the measurement of wound area (wound healing rate was calculated).Wound samples were obtained by excision down to healthy fascia along wound edge.Histological study was done with HE staining.The expression of CD68 (the surface marker of macrophage) in the wound tissue was observed with immunohistochemical staining.The double positive expressions of induced nitric oxide synthase (iNOS) plus CD68 (type Ⅰ macrophage) and arginase 1 (Arg-1)plus CD68 (type Ⅱ macrophage)were observed with immunofluorescence staining.The levels of interferon-γ (IFN-γ),TNF-o,IL-4,IL-13,IL-10,and IL-12 in wound tissue were assayed by double-antibody sandwich ELISA,and the ratio of IL-10/IL-12 was calculated.Full-thickness skin tissues (11 mm diameter) in rats of control group were excised at the same site as rats in injury group,and the histological observation and cytokines assay were performed as well.Data were processed with one-way analysis of variance or LSD-t test.Results Wound area of rats in injury group was gradually reduced after injury,and the overall difference of the wound healing rate on each PID was statistically significant (F =358.55,P <0.01).No abnormal appearance of skin tissue was observed in rats of control group.In injury group,inflammatory cell infiltration was obvious in wound tissue on PID 1 and 3; vascular structure and fresh collagen were observed in wound tissue on PID 7 and 13.Numbers of CD68 positive cells in skin tissue of rats in control group and wound tissue of rats in injury group on PID 1,3,7,and 13 were respectively (2.7±1.5),(31.8 ±3.5),(40.8 ±4