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High urinary homoarginine excretion is associated with low rates of all-cause mortality and graft failure in renal transplant recipients
Tsikas, D. ; Frenay, A.S. ; Kayacelebi, A.A. ; Beckmann, B. ; Soedamah-Muthu, S.S. ; Borst, M.H. de; Berg, E. van den; Bakker, S.J.L. - \ 2015
Amino Acids 47 (2015)9. - ISSN 0939-4451 - p. 1827 - 1836.
glomerular-filtration-rate - nitric-oxide synthase - cardiovascular risk - heart-failure - substrate-specificity - blood-pressure - l-arginine - disease - serum - dysfunction
Renal transplant recipients (RTR) have an increased cardiovascular risk profile. Low levels of circulating homoarginine (hArg) are a novel risk factor for mortality and the progression of atherosclerosis. The kidney is known as a major source of hArg, suggesting that urinary excretion of hArg (UhArg) might be associated with mortality and graft failure in RTR. hArg was quantified by mass spectrometry in 24-h urine samples of 704 RTR (functioning graft =1 year) and 103 healthy subjects. UhArg determinants were identified with multivariable linear regression models. Associations of UhArg with all-cause mortality and graft failure were assessed using multivariable Cox regression analyses. UhArg excretion was significantly lower in RTR compared to healthy controls [1.62 (1.09–2.61) vs. 2.46 (1.65–4.06) µmol/24 h, P <0.001]. In multivariable linear regression models, body surface area, diastolic blood pressure, eGFR, pre-emptive transplantation, serum albumin, albuminuria, urinary excretion of urea and uric acid and use of sirolimus were positively associated with UhArg, while donor age and serum phosphate were inversely associated (model R 2 = 0.43). During follow-up for 3.1 (2.7–3.9) years, 83 (12 %) patients died and 45 (7 %) developed graft failure. UhArg was inversely associated with all-cause mortality [hazard risk (HR) 0.52 (95 % CI 0.40–0.66), P <0.001] and graft failure [HR 0.58 (0.42–0.81), P = 0.001]. These associations remained independent of potential confounders. High UhArg levels are associated with reduced all-cause mortality and graft failure in RTR. Kidney-derived hArg is likely to be of particular importance for proper maintenance of cardiovascular and renal systems.
Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages
Meijerink, J. ; Poland, M.C.R. ; Balvers, M.G.J. ; Plastina, P. ; Lute, C. ; Dwarkasing, J.T. ; Norren, K. van; Witkamp, R.F. - \ 2015
British Journal of Pharmacology 172 (2015)1. - ISSN 0007-1188 - p. 24 - 37.
nitric-oxide synthase - cannabinoid receptor - concise guide - fatty-acids - kappa-b - arrive guidelines - prostaglandin e-2 - fish-oil - anandamide - pharmacology
Background and Purpose N-docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long-chain polyunsaturated n-3 fatty acid docosahexaenoic (DHA; 22: 6n-3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti-inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune-modulating effects of DHEA. Experimental Approach Antagonists for cannabinoid (CB)1 and CB2 receptors and PPAR¿ were used to explore effects of DHEA on NO release by LPS-stimulated RAW264.7 cells. The possible involvement of CB2 receptors was studied by comparing effects in LPS-stimulated peritoneal macrophages obtained from CB2-/- and CB2+/+ mice. Effects on NF-¿B activation were determined using a reporter cell line. To study DHEA effects on COX-2 and lipoxygenase activity, 21 different eicosanoids produced by LPS-stimulated RAW264.7 cells were quantified by LC-MS/MS. Finally, effects on mRNA expression profiles were analysed using gene arrays followed by Ingenuity® Pathways Analysis. Key Results CB1 and CB2 receptors or PPARs were not involved in the effects of DHEA on NO release. NF-¿B and IFN-ß, key elements of the myeloid differentiation primary response protein D88 (MyD88)-dependent and MyD88-independent pathways were not decreased. By contrast, DHEA significantly reduced levels of several COX-2-derived eicosanoids. Gene expression analysis provided support for an effect on COX–2-mediated pathways. Conclusions and Implications Our findings suggest that the anti-inflammatory effects of DHEA in macrophages predominantly take place via inhibition of eicosanoids produced through COX-2.
Angiopoietin-like 4 Stimulates STAT3-mediated iNOS Expression and Enhances Angiogenesis to Accelerate Wound Healing in Diabetic Mice
Chong, H.C. ; Goh, C.Q. ; Gounko, N.V. ; Luo, B. ; Wang, X. ; Kersten, A.H. - \ 2014
Molecular Therapy 22 (2014)9. - ISSN 1525-0016 - p. 1593 - 1604.
nitric-oxide synthase - solid human tumors - international consensus - growth-factors - foot ulcers - repair - cells - quantification - methodology - mechanisms
Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.
Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work?
Kromhout, D. ; Yasuda, S. ; Geleijnse, J.M. ; Shimokawa, H. - \ 2012
European Heart Journal 33 (2012)4. - ISSN 0195-668X - p. 436 - 443.
coronary-heart-disease - polyunsaturated fatty-acids - sudden cardiac death - placebo-controlled trial - endothelium-dependent responses - randomized controlled-trials - nitric-oxide synthase - long-term treatment - cod-liver oil - eicosapentaenoic acid
Omega-3 fatty acids, which are found abundantly in fish oil, exert pleiotropic cardiometabolic effects with a diverse range of actions. The results of previous studies raised a lot of interest in the role of fish oil and omega-3 fatty acids in primary and secondary prevention of cardiovascular diseases. The present review will focus on the current clinical uses of omega-3 fatty acids and provide an update on their effects. Since recently published trials in patients with coronary artery diseases or post-myocardial infarction did not show an effect of omega-3 fatty acids on major cardiovascular endpoints, this review will examine the limitations of those data and suggest recommendations for the use of omega-3 fatty acids
Heterogeneity of macrophage activation in fish
Forlenza, M. ; Fink, I.R. ; Raes, G. ; Wiegertjes, G.F. - \ 2011
Developmental and Comparative Immunology 35 (2011)12. - ISSN 0145-305X - p. 1246 - 1255.
tumor-necrosis-factor - cyprinus-carpio l. - trout oncorhynchus-mykiss - nitric-oxide synthase - toll-like receptors - interferon-gamma genes - carassius-auratus l. - expression analysis - rainbow-trout - factor-alpha
In this review, we focus on four different activation states of fish macrophages. In vitro, stimulation with microbial ligands induces the development of innate activated macrophages whereas classically activated macrophages can be induced by stimulation with LPS in combination with (recombinant) IFN¿. Both types of macrophages show elevated phagocytic activity, expression of pro-inflammatory cytokine genes and radical production. Alternatively activated macrophages require the cytokines IL-4/IL-13 for induction of, among others, arginase activity. Until in vitro studies identify the effects of putative IL-4 and IL-13 homologues on fish macrophages, arginase enzyme activity remains the most reliable marker for the presence of alternatively activated macrophages in fish. The best evidence for the existence of regulatory macrophages, associated with the presence of IL-10, comes from in vivo studies, for example during parasitic infections of carp. Altogether, differentially activated macrophages in fish largely resemble the phenotypes of mammalian macrophages. However, the presence of fish-specific ligand recognition by TLRs and of duplicated genes coding for proteins with particular activities, poses additional challenges for the characterization of phenotype-specific gene signatures and cell surface markers.
The ethanolamide metabolite of DHA, docosahexaenoylethanolamine, shows immunomodulating effects in mouse peritoneal and RAW264.7 macrophages: evidence for a new link between fish oil and inflammation
Meijerink, J. ; Plastina, P. ; Vincken, J.P. ; Poland, M.C.R. ; Attya, M. ; Balvers, M.G.J. ; Gruppen, H. ; Gabriele, B. ; Witkamp, R.F. - \ 2011
British Journal of Nutrition 105 (2011)12. - ISSN 0007-1145 - p. 1798 - 1807.
nitric-oxide synthase - monocyte chemoattractant protein-1 - fatty-acid-composition - docosahexaenoic acid - n-acylethanolamine - insulin-resistance - adipose-tissue - reduces atherosclerosis - eicosapentaenoic acid - dietary-fat
Several mechanisms have been proposed for the positive health effects associated with dietary consumption of long-chain n-3 PUFA (n-3 LC-PUFA) including DHA (22 : 6n-3) and EPA (20 : 5n-3). After dietary intake, LC-PUFA are incorporated into membranes and can be converted to their corresponding N-acylethanolamines (NAE). However, little is known on the biological role of these metabolites. In the present study, we tested a series of unsaturated NAE on the lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages. Among the compounds tested, docosahexaenoylethanolamine (DHEA), the ethanolamide of DHA, was found to be the most potent inhibitor, inducing a dose-dependent inhibition of NO release. Immune-modulating properties of DHEA were further studied in the same cell line, demonstrating that DHEA significantly suppressed the production of monocyte chemotactic protein-1 (MCP-1), a cytokine playing a pivotal role in chronic inflammation. In LPS-stimulated mouse peritoneal macrophages, DHEA also reduced MCP-1 and NO production. Furthermore, inhibition was also found to take place at a transcriptional level, as gene expression of MCP-1 and inducible NO synthase was inhibited by DHEA. To summarise, in the present study, we showed that DHEA, a DHA-derived NAE metabolite, modulates inflammation by reducing MCP-1 and NO production and expression. These results provide new leads in molecular mechanisms by which DHA can modulate inflammatory processes.
Beta-carotene affects gene-expression in lungs of male and female Bcmo1-/-mice in opposite directions
Helden, Y.G.J. ; Godschalk, R.W.L. ; Swarts, J.J.M. ; Hollman, P.C.H. ; Schooten, F.J. van; Keijer, J. - \ 2011
Cellular and Molecular Life Sciences 68 (2011)3. - ISSN 1420-682X - p. 489 - 504.
nitric-oxide synthase - base-line characteristics - retinol efficacy trial - vitamin-a - cardiovascular-disease - epidemiologic evidence - cancer incidence - estrous-cycle - double-tracer - women
Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,150-monooxygenase 1 knockout (Bcmo1-/-) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1-/- mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1-/- mice. Testosterone levels were higher after BC supplementation only in Bcmo1-/- mice, which had, unlike wild-type (Bcmo1?/?) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.
Comparative Study of the Ability of Leishmania mexicana Promastigotes and Amastigotes To Alter Macrophage Signaling and Functions
Abu-Dayyeh, I. ; Hassani, K. ; Westra, E.R. ; Mottram, J.C. ; Olivier, M. - \ 2010
Infection and Immunity 78 (2010)6. - ISSN 0019-9567 - p. 2438 - 2445.
nitric-oxide synthase - tyrosine-phosphatase shp-1 - nf-kappa-b - interferon-gamma - murine leishmaniasis - cysteine peptidases - virulence factors - protein - gene - expression
Leishmania alternates between two morphologically different stages, promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage (M phi) signaling and function, fewer reports investigated signaling alterations mediated by amastigotes, and there is a lack of comparative studies. In this study, we performed a comparison between the ability of both forms of the parasite to alter M phi signaling and functions. Here, we show that both promastigotes and amastigotes were able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and that lmcpb(-/-) promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1 alpha and AP-1, but we show differences in the modulation of NF-kappa B, with promastigotes cleaving the p65 subunit, generating a smaller p35 subunit, and amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-kappa B, STAT-1 alpha, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to gamma interferon (IFN-gamma) stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-gamma-mediated nitric oxide (NO) production in M phi s.
Functional analysis of carp interferon-y: Evolutionary conservation of classical phagocyte acativation
Arts, J.A.J. ; Tijhaar, E. ; Chadzinska, M.K. ; Savelkoul, H.F.J. ; Verburg-van Kemenade, B.M.L. - \ 2010
Fish and Shellfish Immunology 29 (2010)5. - ISSN 1050-4648 - p. 793 - 802.
nitric-oxide synthase - common carp - ifn-gamma - molecular characterization - expression analysis - messenger-rna - tnf-alpha - nf-kb - macrophages - l.
In teleost fish two IFN-¿ gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-¿ function is associated with the IFN-¿2 cluster. We investigated the evolutionary conservation of the IFN-¿ function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-¿ sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-¿2, in contrast to carp IFN-¿1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1ß, TNFa and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-¿ sensitive. These results corroborate an evolutionary conserved nature of IFN-¿ function in lower vertebrates including classical activation of phagocytes
Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds
Chanput, W. ; Mes, J.J. ; Vreeburg, R.A.M. ; Savelkoul, H.F.J. ; Wichers, H.J. - \ 2010
Food & Function 1 (2010)3. - ISSN 2042-6496 - p. 254 - 261.
nf-kappa-b - necrosis-factor-alpha - blood mononuclear-cells - nitric-oxide synthase - gene-expression - messenger-rna - signaling pathways - cytokine production - citrus pectin - tnf-alpha
An assay was developed to study inflammation-related immune responses of food compounds on monocytes and macrophages derived from THP-1 cell line. First strategy focused on the effects after stimulation with either lipopolysaccharide (LPS) or Concanavalin A (ConA). Gene expression kinetics of inflammation-related cytokines (IL-1ß, IL-6, IL-8, IL-10 and TNF-a), inflammation-related enzymes (iNOS and COX-2), and transcription factors (NF-¿B, AP-1 and SP-1) were analyzed using RT-PCR. Time dependent cytokine secretion was investigated to study the inflammation-related responses at protein level. LPS stimulation induced inflammation-related cytokine, COX-2 and NF-¿B genes of THP-1 monocytes and THP-1 macrophages with the maximum up-regulation at 3 and 6 h, respectively. These time points, were subsequently selected to investigate inflammation modulating activity of three well known immuno-modulating food-derived compounds; quercetin, citrus pectin and barley glucan. Co-stimulation of LPS with either quercetin, citrus pectin, or barley glucan in THP-1 monocytes and macrophages showed different immuno-modulatory activity of these compounds. Therefore, we propose that simultaneously exposing THP-1 cells to LPS and food compounds, combined with gene expression response analysis are a promising in vitro screening tool to select, in a limited time frame, food compounds for inflammation modulating effects.
Long-term wine consumption is related to cardiovascular mortality and life expectancy independently of moderate alcohol intake: the Zutphen Study
Streppel, M.T. ; Ocke, M.C. ; Boshuizen, H.C. ; Kok, F.J. ; Kromhout, D. - \ 2009
Journal of Epidemiology and Community Health 63 (2009)7. - ISSN 0143-005X - p. 534 - 540.
coronary-heart-disease - nitric-oxide synthase - us male physicians - middle-aged men - follow-up - all-cause - red wine - beverage preference - japanese men - ex-drinkers
Background: Light to moderate alcohol intake lowers the risk of cardiovascular mortality, but whether this protective effect can be attributed to a specific type of beverage remains unclear. Moreover, little is known about the effects of long-term alcohol intake on life expectancy. Methods: The impact of long-term alcohol intake and types of alcoholic beverages consumed on cardiovascular mortality and life expectancy at age 50 was investigated in the Zutphen Study, a cohort of 1373 men born between 1900 and 1920 and examined repeatedly between 1960 and 2000. Hazard ratios (HRs) for total alcohol intake and alcohol from wine, beer and spirits were obtained from time-dependent Cox regression models. Life expectancy at age 50 was calculated from areas under survival curves. Results: Long-term light alcohol intake, that is =20 g per day, compared with no alcohol, was strongly and inversely associated with cerebrovascular (HR 0.43, 95% CI 0.26 to 0.70), total cardiovascular (HR 0.70, 95% CI 0.55 to 0.89) and all-cause mortality (HR 0.75, 95% CI 0.63 to 0.91). Independent of total alcohol intake, long-term wine consumption of, on average, less than half a glass per day was strongly and inversely associated with coronary heart disease (HR 0.61, 95% CI 0.41 to 0.89), total cardiovascular (HR 0.68, 95% CI 0.53 to 0.86) and all-cause mortality (HR 0.73, 95% CI 0.62 to 0.87). These results could not be explained by differences in socioeconomic status. Life expectancy was about 5 years longer in men who consumed wine compared with those who did not use alcoholic beverages. Conclusion: Long-term light alcohol intake lowered cardiovascular and all-cause mortality risk and increased life expectancy. Light wine consumption was associated with 5 years longer life expectancy; however, more studies are needed to verify this result
Differential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model
Forlenza, M. ; Taverne-Thiele, J.J. ; Kachamakova, N. ; Scharsack, J.P. ; Rombout, J.H.W.M. ; Wiegertjes, G.F. - \ 2008
Molecular Immunology 45 (2008)11. - ISSN 0161-5890 - p. 3178 - 3189.
carp cyprinus-carpio - nitric-oxide synthase - trypanoplasma-borreli protozoa - protein-tyrosine nitration - in-vivo - functional-characterization - lymphocyte-activation - glutathione-reductase - trypanosoma-carassii - murine macrophages
Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host¿parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host¿parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.
Differential macrophage polarisation during parasitic infections in common carp (Cyprinus carpio L.)
Joerink, M. ; Forlenza, M. ; Ribeiro, C.M.S. ; Vries, B.J. de; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2006
Fish and Shellfish Immunology 21 (2006)5. - ISSN 1050-4648 - p. 561 - 571.
nitric-oxide synthase - goldfish kidney leukocytes - trypanoplasma-borreli - functional-characterization - alternative activation - leishmania-major - immune-response - cell-line - blood - arginase
In many parasitic infections both classically activated macrophages (caMF) and alternatively activated macrophages (aaMF) play a pivotal role. To investigate if both types of macrophages also play an important role during parasitic infections in fish, we infected carp with either Trypanoplasma borreli or Trypanosoma carassii and determined the activation state of the head kidney leukocytes (HKL). Nitrite production was used as read-out for caMF and arginase activity as read-out for aaMF. Basal nitrite production and arginase activity of HKL were moderately different between the two infections. Differences were observed, however, after ex vivo re-stimulation of HKL. Re-stimulation with LPS and T. borreli lysates increased nitrite production by HKL of T. borreli-infected fish. Re-stimulation with cAMP increased arginase activity in HKL of T. carassii-infected fish. Our results indicate that T. borreli-infected carp are more prone to increase nitrite production by caMF while T. carassii-infected fish are more prone to increase arginase activity by aaMF
Head kidney-derived macrophages of common carp (Cyprinus carpio L.) show plasticity and functional polarization upon differential stimulation
Joerink, M. ; Ribeiro, C.M.S. ; Stet, R.J.M. ; Hermsen, G.J. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2006
The Journal of Immunology 177 (2006)1. - ISSN 0022-1767 - p. 61 - 69.
nitric-oxide synthase - alternatively activated macrophages - trout oncorhynchus-mykiss - cell-line - in-vitro - teleost macrophages - respiratory burst - expression - fish - receptor
Cells from the myeloid lineage are pluripotent, To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we plienotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synihase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish
Evolutionary conservation of alternative activation of macrophages: structural and functional characterization of arginase 1 and 2 in carp (Cyprinus carpio L.)
Joerink, M. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2006
Molecular Immunology 43 (2006)8. - ISSN 0161-5890 - p. 1116 - 1128.
nitric-oxide synthase - goldfish kidney leukocytes - hydroxy-l-arginine - cell-line - differential regulation - murine macrophages - rainbow-trout - messenger-rna - teleost fish - expression
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 for teleosts. Arginase is the characteristic enzyme in aaMF and two isoforms have been described for mammals. To study the presence of aaMF in a primitive vertebrate species we cloned arginase 1 and 2 cDNA of common carp. Carp arginase 1 is a 340 aa protein with 63% aa sequence identity to human arginase 1. Carp arginase 2 is a 347 aa protein with 63% aa sequence identity to human arginase 2. Three highly homologous arginase 2 genes were found, each showing only single non-synonymous substitutions. Basal arginase 1 expression is mainly found in carp mid kidney. In contrast, arginase 2 was expressed in all organs examined with the highest basal gene expression in liver. Cultured carp head kidney-derived macrophages were used to study aaMF in vitro. Carp macrophages showed significant arginase activity which could be induced by dibutyryl cyclic adenosine mono phosphate (cAMP) and specifically inhibited by NG-hydroxy-l-arginine (NOHA). At the gene level, arginase 2 gene expression was upregulated by cAMP stimulation, while arginase 1 gene expression was not influenced. LPS stimulation did not alter the arginase 1 or 2 expression, inducible nitric oxide synthase (iNOS) expression was, however, upregulated. This expression of iNOS was used as a measure of classical activation of carp macrophages. Thus, in contrast to mammals, fish arginase 2 and not arginase 1 is differentially regulated and likely involved in the alternative activation of fish macrophages. Our data suggest there may be an evolutionary conservation of the presence of aaMF down to teleost fish.
Phosphodiesterase type 5 inhibition improves early memory consolidation of object information
Prickaerts, J. ; Sik, A. ; Staveren, W.C.G. ; Koopmans, G. ; Steinbusch, H.W.M. ; Staay, F.J. van der; Vente, J. de; Blokland, A. - \ 2004
Neurochemistry International 45 (2004)6. - ISSN 0197-0186 - p. 915 - 928.
nitric-oxide synthase - long-term potentiation - dependent protein-kinase - cultured hippocampal-neurons - soluble guanylyl cyclase - cerebral-blood-flow - c-fos expression - 14-unit t-maze - late-phase ltp - cyclic-gmp
The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.
Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica
Baarlen, P. van; Staats, M. ; Kan, J.A.L. van - \ 2004
Molecular Plant Pathology 5 (2004)6. - ISSN 1464-6722 - p. 559 - 574.
pyrenophora-tritici-repentis - disease-resistance response - host-selective toxins - nitric-oxide synthase - hypersensitive response - induced apoptosis - defense response - possible roles - cytochrome-c - plant-cells
The genus Botrytis contains necrotrophic plant pathogens that have a wide host range (B. cinerea) or are specialized on a single host species, e.g. B. elliptica on lily. In this study, it was found that B. elliptica-induced cell death of lily displays hallmark features of animal programmed cell death or apoptosis including cytoplasmic shrinkage, nuclear DNA fragmentation and the accumulation of NO as well as H2O2. A pharmacological approach showed that B. elliptica-induced cell death could be modulated by serine and cysteine protease inhibitors including one caspase inhibitor. Blocking phosphatase activity stimulated cell death and concomitant lesion formation, suggesting that B. elliptica-induced cell death is mediated by kinase/phosphatase pathways. Blocking Ca2+ influx restricted cell death. Blocking steps of sphingolipid biosynthesis delayed lily cell death for several days. B. elliptica culture filtrate (CF) was able to induce lily cell death by means of secreted proteins. Induction of cell death is necessary and sufficient for pathogenicity and host specialization because prior infiltration of B. elliptica CF enabled subsequent infection of lily by the otherwise incompatible pathogens B. cinerea and B. tulipae. The secreted B. elliptica proteins also induced cell death in some but not all Arabidopsis accessions and mutants. Arabidopsis accessions that respond to infiltration of B. elliptica CF also display cell death symptoms upon inoculation with B. elliptica conidia.
Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2
Stierum, R. ; Gaspari, M. ; Dommels, Y.E.M. ; Ouatas, T. ; Pluk, H. ; Jespersen, S. ; Vogels, J. ; Verhoeckx, K. ; Groten, J. ; Ommen, B. van - \ 2003
Biochimica et Biophysica Acta. Proteins & Proteomics 1650 (2003)1-2. - ISSN 1570-9639 - p. 73 - 91.
creatine-kinase bb - glutathione s-transferases - alzheimers-disease brain - nitric-oxide synthase - acid-binding-protein - human colon-cancer - down-regulation - 1,25-dihydroxyvitamin d-3 - gene-expression - actin dynamics
Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: ¿3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4¿7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of ¿-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase ¿ (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.