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Macrophage Cell Line (macrophage + cell_line)
Kinds of Macrophage Cell Line Selected AbstractsTargeted replacement of rodent CCR2 with the human orthologue CCR2B: A mouse model for in vivo analysis of human target-selective small molecule MCP-1 receptor antagonistsDRUG DEVELOPMENT RESEARCH, Issue 4 2002Haydn M. Prosser Abstract Rodent models for testing the efficacy of lead compounds are often invalidated by species selectivity of the compounds. The advent of mouse embryonic stem cell technology has allowed the development of genetically engineered mouse strains that incorporate a specific human gene in place of the orthologous mouse gene, a so-called knock-in mouse. This study describes the generation and validation of a mutant mouse line that expresses human CCR2B as a functional substitute for murine CCR2. The human CCR2B knock-in mice are viable and appear normal. In vitro assays indicate that the CCR2B knock-in is functionally expressed, giving a macrophage chemotactic profile in response to JE or MCP-1 that is similar to human peripheral blood monocytes rather than that of a murine macrophage cell line. In addition, the human selective CCR2B antagonist, SB-399721, was a more potent inhibitor of CCR2B knock-in macrophages in response to hMCP-1 than JE. The ability of the human CCR2B gene to functionally substitute for the mouse orthologue in vivo is demonstrated by a normal inflammatory response to intraperitoneal thioglycollate injection. Drug Dev. Res. 55:197,209, 2002. © 2002 Wiley-Liss, Inc. [source] Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophagesFEBS JOURNAL, Issue 6 2007Bronwyn E. Brown Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glucose or metabolic processes) to low-density lipoproteins has been previously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atherosclerosis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been characterized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester levels were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was significantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Differences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumulation of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls. [source] Interferon-, and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophagesFEBS JOURNAL, Issue 22 2000S. L. Wardrop The natural resistance associated macrophage protein 2 (Nramp2) is a transporter that is involved in iron (Fe) uptake from transferrin (Tf) and low molecular mass Fe complexes. Here we describe the effect of the inflammatory mediators interferon-, (IFN-,) and lipopolysaccharide (LPS) on the expression of Nramp2 mRNA and Fe uptake by cells of the macrophage lineage. After incubation of the RAW264.7 macrophage cell line with LPS there was a sevenfold increase in the expression of the 2.3 kb Nramp2 mRNA transcript when compared with the control, but little effect on the Nramp2 3.1 kb transcript. These results indicate differential regulation of the two transcripts. Treatment with LPS resulted in an increase in 59Fe uptake from 59Fe,nitrilotriacetic acid, while transferrin receptor (TfR) mRNA levels and 59Fe uptake from 59Fe,Tf were decreased. Paradoxically, at the same time, an increase in iron regulatory protein (IRP)1 RNA-binding activity was observed. Incubation with IFN-, (50 U·mL,1) resulted in a marked decrease in TfR mRNA levels but had no effect on Nramp2 mRNA expression. Exposure of RAW264.7 cells to both IFN-, and LPS resulted in a fourfold increase in the Nramp2 2.3-kb transcript and a four to fivefold decrease in the 3.1-kb transcript when compared with the control. Furthermore, there was a decrease in TfR mRNA levels despite an increase in IRP1 RNA-binding activity and a marked increase in inducible nitric oxide synthase mRNA expression. Hence, TfR and Nramp2 mRNA expression did not appear to be regulated in a concerted manner. Similar responses to those found above for RAW264.7 cells were also observed in the J774 macrophage cell line and also for primary cultures of mouse peritoneal macrophages. These results are of interest as the TfR and Nramp2 are thought to act together during Fe uptake from Tf. This is the first report to demonstrate regulation of the Nramp2 mRNA transcripts by inflammatory mediators. [source] Bone morphogenetic protein-6 induces the expression of inducible nitric oxide synthase in macrophagesIMMUNOLOGY, Issue 1pt2 2009Seok J. Kwon Summary Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-, (TGF-,) superfamily. In the present study, we investigated the effect of BMPs on the production of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line, RAW 264.7, and in mouse peritoneal macrophages. Among the BMPs, only BMP-6 induced iNOS expression in a time-dependent and dose-dependent manner in both cell types. Induction of iNOS was inhibited by both cycloheximide and actinomycin D, indicating that the induction of iNOS expression by BMP-6 requires new protein synthesis. Mechanistic studies revealed that the BMP-6-induced iNOS expression requires both Smads and nuclear factor-kappa B (NF-,B) signalling pathways. Furthermore, induction of interleukin-1, (IL-1,) was necessary for iNOS induction by BMP-6. These observations suggest that BMP-6 stimulates macrophages to produce iNOS through IL-1, via Smad and NF-,B signalling pathways and that BMP-6 may be an important regulator of macrophages. [source] Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophagesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Yen-Chou Chen Abstract Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N -nitro- L -arginine (NLA) or N -nitro- L -arginine methyl ester (L -NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L -arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L -NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L -NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L -arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes. J. Cell. Biochem. 82: 537,548, 2001. © 2001 Wiley-Liss, Inc. [source] Lycopene Inhibits LPS-Induced Proinflammatory Mediator Inducible Nitric Oxide Synthase in Mouse Macrophage CellsJOURNAL OF FOOD SCIENCE, Issue 1 2007Mohamed M. Rafi ABSTRACT:, Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 ,M) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. [source] An extract of Apium graveolens var. dulce leaves: structure of the major constituent, apiin, and its anti-inflammatory propertiesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2007T. Mencherini Flavonoids, natural compounds widely distributed in the plant kingdom, are reported to affect the inflammatory process and to possess anti-inflammatory as well as immunomodulatory activity in-vitro and in-vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of the ethanol/water (1:1) extract of the leaves of Apium graveolens var. dulce (celery) on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated for 24 h with Escherichia coli lipopolysaccharide (LPS) were evaluated. The extract of A. graveolens var. dulce contained apiin as the major constituent (1.12%, w/w, of the extract). The extract and apiin showed significant inhibitory activity on nitrite (NO) production in-vitro (IC50 0.073 and 0.08 mg mL,1 for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL,1 for the extract and apiin, respectively) in LPS-activated J774.A1 cells. The croton-oil ear test on mice showed that the extract exerted anti-inflammatory activity in-vivo (ID50 730 ,g cm,2), with a potency seven-times lower than that of indometacin (ID50 93 ,g cm,2), the non-steroidal anti-inflammatory drug used as reference. Our results clearly indicated the inhibitory activity of the extract and apiin in-vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. The anti-inflammatory properties of the extract demonstrated in-vivo might have been due to reduction of iNOS enzyme expression. [source] Effects of phenylethanoid glycosides from Digitalis purpurea L. on the expression of inducible nitric oxide synthaseJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2005Jae Wook Oh We have isolated four different phenylethanoid glycosides (purpureaside A, desrhamnosyl acteo-side, calceolarioside B and plantainoside D) from the leaves of Digitalis purpurea (foxglove). The effects of these glycosides on activator protein-1 (AP-1)-mediated inducible nitric oxide synthase (iNOS) gene expression in the Raw264.7 macrophage cell line have been studied. Of these four glycosides, purpureaside A potently inhibited iNOS induction by lipopolysaccharide (LPS). Increase in iNOS mRNA by LPS was completely suppressed by purpureaside A. Purpureaside A did not significantly affect LPS-inducible nuclear factor- kB (NF- kB) activation or the nuclear translocation of p65. Moreover, a reporter gene assay using AP-1 specific luciferase reporter revealed that the enhanced activity of AP-1 by LPS was completely abolished in cells treated with purpureaside A. These results demonstrated that purpureaside A inhibited LPS-inducible iNOS expression in macrophages through the suppression of AP-1, but not of NF- kB. [source] The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to EndotoxinALCOHOLISM, Issue 2004Nobuyuki Enomoto Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-, (TNF-,) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-, should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca2+]i) is required for LPS-induced TNF-, production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca2+]i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin. Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 ,mol/liter). After addition of LPS (10 ,g/ml) to culture media, [Ca2+]i was measured using a fluorescent indicator, fura-2. Results: LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 ,g/ml)-induced TNF-, production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05). Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin. [source] A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cellsMICROBIOLOGY AND IMMUNOLOGY, Issue 10 2009Mari Nomoto ABSTRACT The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc2155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes. [source] Effect of pistachio oil on gene expression of IFN-induced protein with tetratricopeptide repeats 2: A biomarker of inflammatory responseMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S1 2010Jun Zhang Abstract When incorporated into the diet, pistachios have a beneficial effect on lipid and lipoprotein profiles. However, little is known about potential anti-inflammatory properties. This study was conducted to determine whether pistachio oil and an organic extract from pistachio oil extract (PE) regulated expression of inflammation-related genes. A mouse macrophage cell line (RAW 264.7 cells) was treated with pistachio oil and gene expression microarray analyses were performed. Pistachio oil significantly affected genes involved in immune response, defense response to bacteria, and gene silencing, of which INF-induced protein with tetratricopeptide repeats 2 (Ifit-2) was the most dramatically reduced. PE reduced the LPS-induced Ifit-2 by 78% and the bioactive molecules contained in PE, linoleic acid, and ,-sitosterol recapitulated this inhibition. Promoter analysis identified two adjacent IFN-stimulated response elements, which lie between ,110 and ,85bp of the 5,-flanking region of the Ifit-2 promoter, as being responsive to LPS activation and inhibition by PE. Our results indicate that pistachio oil and bioactive molecules present therein decrease Ifit-2 expressions, and due to the sensitivity of this effect, this gene is a potential biomarker for monitoring diet-induced changes in inflammation. [source] The role of cyclic-AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 6 2006W. Sosroseno Aims:, The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods:, The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results:, Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion:, The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway. [source] Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 3 2006W. Sosroseno Aims:, The aim of the present study was to determine whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells). Methods:, RAW264.7 cells were treated with A. actinomycetemcomitans- lipopolysaccharide or lipopolysaccharide from Escherichia coli for 24 h. The effect of polymyxin B, l -norvaline, dl -norvaline, dexamethasone and cytokines (interferon-, and interleukin-4) on arginase activity in A. actinomycetemcomitans- lipopolysaccharide-stimulated cells was also determined. The cells were pretreated with anti-CD14, anti -toll-like receptor 2, or anti-toll-like receptor 4 antibody prior to stimulation with A. actinomycetemcomitans- lipopolysaccharide. Arginase activity was determined by a colorimetric assay. Results:,A. actinomycetemcomitans- lipopolysaccharide stimulated arginase activity in RAW264.7 cells in a dose-dependent manner, but was less potent than E. coli- lipopolysaccharide. Polymyxin B and l -norvaline, but not dl -norvaline, blocked the arginase activity in A. actinomycetemcomitans- lipopolysaccharide-stimulated cells. Dexamethasone and interleukin-4 but not interferon-, augmented arginase activity in A. actinomycetemcomitans- lipopolysaccharide-stimulated cells. Treatment of the cells with anti-CD14 and anti-toll-like receptor 4 but not anti-toll-like receptor 2 antibody decreased arginase activity in A. actinomycetemcomitans- lipopolysaccharide-stimulated cells. Conclusion:, The results of the present study suggest that lipopolysaccharide from A. actinomycetemcomitans via CD14/toll-like receptor 4 complex molecules and the regulatory control of glucocorticoid and cytokines may stimulate arginase activity in RAW264.7 cells. [source] Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 2 2002W. Sosroseno The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS- A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS- A. actinomycetemcomitans or Escherichia coli LPS (LPS- Ec) for 24 h. The effects of NG -monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-,, TNF-,, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS- A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS- A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS- Ec. NMMA and polymyxin B blocked the production of NO. IFN-, and IL-12 potentiated but IL-4 depressed NO production by LPS- A. actinomycetemcomitans -stimulated RAW264.7 cells. TNF-, had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages. [source] Release of (1,3)-,-D-Glucan from Depth-type Membrane Filters and Their In Vitro Effects on Proinflammatory Cytokine ProductionARTIFICIAL ORGANS, Issue 8 2003Atsushi Ohata Abstract:, To clarify the origin of (1,3)-,-D-glucan in blood products and assess the biological activity of filter extracts, we evaluated (1,3)-,-D-glucan extraction from depth filters used to process blood products and their in vitro effects on proinflammatory cytokine production from macrophages. Cellulose or nylon filters were analyzed for (1,3)-,-D-glucan using the Fungitec G test. To evaluate the biological activity of the filter extracts, Mono Mac 6 cells (a human macrophage cell line) were cultured with filter extracts with or without lipopolysaccharide, and tumor necrosis factor-alpha (TNF-,) and interleukin-1 beta (IL-1,) concentrations in the culture media were measured. (1,3)-,-D-Glucan was released from seven cellulose filters but the nylon filter level was undetectable. Proinflammatory cytokine production ranged from 74.3% to 119.0% of the control for TNF-, and 81.2% to 115.9% for IL-1,. TNF-, and IL-1, levels were low without lipopolysaccharide. The data indicate that (1,3)-,-D-glucan in blood products is contaminated with the depth filters and that these filter extracts modulate proinflammatory cytokine production from macrophages. [source] Anticancer activity of hydrophobic peptides from soy proteinsBIOFACTORS, Issue 1-4 2000Song E Kim Abstract An anticancer peptide from soy protein was purified and isolated. Defatted soy protein was hydrolyzed with thermoase and hydrophobic peptides were extracted with ethanol. The peptide extract was fractionated by XAD-2 hydrophobic, gel filtration chromatography, and different C18 HPLCs. Anticancer activity of each fraction was assayed by measuring in vitro cytotoxicity on P388D1, a mouse monocyte macrophage cell line. IC50 value of a peptide fraction from Sephadex G-25 chromatography was 0.16 mg/ml. This peptide fraction at 1 mg/ml significantly affected cell cycle progression by arresting P388D1 at G2/M phases. Finally purified peptide from analytical C18 HPLC was nonapeptide of which molecular weight was 1157 Da and the sequence was X-Met-Leu-Pro-Ser-Tye-Ser-Pro-Tyr. [source] Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation systemCELLULAR MICROBIOLOGY, Issue 2 2006Chris A. L. Oura Summary The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re-expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin-like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin-like substrate, bISG15, were expressed at a low level in T. annulata -infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2 -transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells. [source] A Macrophage Cell Model for Selective Metalloproteinase Inhibitor DesignCHEMBIOCHEM, Issue 13 2008Faith E. Jacobsen Abstract The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over the course of the last 30 years, led to the development of a plethora of MMP inhibitors that bind directly to the active-site metal. With one exception, all of these drugs have failed in clinical trials, due to many factors, including an apparent lack of specificity for MMPs. To address the question of whether these inhibitors are selective for MMPs in a biological setting, a cell-based screening method is presented to compare the relative activities of zinc, heme iron, and non-heme iron enzymes in the presence of these compounds using the RAW264.7 macrophage cell line. We screened nine different zinc-binding groups (ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors developed in our laboratory to determine their selectivities against five different metalloenzymes. Using this model, we identified two nitrogen donor compounds,2,2,-dipyridylamine (DPA) and triazacyclononane (TACN),as the most selective ZBGs for zinc metalloenzyme inhibitor development. We also demonstrated that the model could predict known nonspecific interactions of some of the most commonly used MMPis, and could also give cross-reactivity information for newly developed MMPis. This work demonstrates the utility of cell-based assays in both the design and the screening of novel metalloenzyme inhibitors. [source] Use of Selenium to Detect Mercury in Water and Cells: An Enhancement of the Sensitivity and Specificity of a Seleno Fluorescent ProbeCHEMISTRY - A EUROPEAN JOURNAL, Issue 13 2009Bo Tang Prof. Abstract Seleno fluorescent probe: An organoselenium fluorescent probe (FSe-1) for mercury was designed based on the irreversible deselenation mechanism. FSe-1 exhibits an ultrahigh selectivity and sensitivity for Hg2+ detection only for reactive selenium atom sites, due the strong affinity between Se and Hg. Furthermore, the new probe has been successfully used for imaging mercury ions in RAW,264.7 cells (a mouse macrophage cell line; see figure). Inspired by the antitoxic function of selenium towards heavy-metal ions, we designed an organoselenium fluorescent probe (FSe-1) for mercury. The reaction of FSe-1 and Hg2+ is an irreversible deselenation mechanism based on the selenophilic character of mercury. FSe-1 exhibits an ultrahigh selectivity and sensitivity for Hg2+ detection only for reactive selenium atom sites due to the strong affinity between Se and Hg. The experimental results proved that FSe-1 was selective for Hg2+ ions over other relevant metal ions and bioanalytes, and also showed an enhancement in sensitivity of up to 1.0,nM, which is lower than the current Environmental Protection Agency standard for drinking water. Furthermore, the new probe has been successfully applied to the imaging of mercury ions in RAW,264.7 cells (a mouse macrophage cell line) with high sensitivity and selectivity. [source] Surfactant protein D inhibits mite-induced alveolar macrophage and dendritic cell activations through TLR signalling and DC-SIGN expressionCLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2010C-F Liu Summary Background Surfactant protein D (SP-D), a secreted pattern recognition molecule associated with pulmonary innate immunity, has been shown to mediate the clearance of pathogens in multiple ways. However, how SP-D interacts with alveolar macrophages (AMs) and dendritic cells (DCs) during allergen exposure remains unclear. Objective This study was performed to characterize the immunomodulatory effects of SP-D on mite allergen (Dermatophagoides pteronyssinus, Der p)-induced inflammatory signalling in AMs and DCs. Methods Murine AM, alveolar macrophage cell line derived from BALB/c mice (MH-S cells), and human monocyte-derived dendritic cells (MDDC) were used as model systems. The production of nitric oxide (NO) and TNF-,, expression of surface Toll-like receptors (TLRs), and expression of the C-type lectin receptor known as dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN) were measured as a function of pretreatment with SP-D and subsequent exposure to Der p. Der p-dependent cellular activations that were modified by SP-D in these model systems were then identified. Results Pretreatment of MH-S cells with SP-D reduced Der p-dependent production of NO, TNF-,, and the downstream activations of IL-1 receptor-associated kinase, mitogen activated protein kinase (MAPK) kinase, and nuclear factor-,B. SP-D interacted with CD14 such that CD14 binding to Der p was inhibited and Der p-induced signalling via TLRs was blocked. DC-SIGN expression was suppressed by Der p in MH-S and MDDC; this down-regulation of DC-SIGN expression was prevented by pretreatment with SP-D. Conclusions These results indicated that the inhibition of Der p-induced activation of MH-S and MDDC by SP-D is mediated through suppression of the CD14/TLR signalling pathway and maintenance of DC-SIGN expression, which may protect allergen-induced airway inflammation. Cite this as: C-F Liu, M. Rivere, H-J Huang, G. Puzo and J-Y Wang, Clinical & Experimental Allergy, 2010 (40) 111,122. [source] Comparative Evaluation of Cytokines, T-Cell Apoptosis, and Costimulatory Molecule Expression in Tuberculous and Nontuberculous PleurisyCLINICAL AND TRANSLATIONAL SCIENCE, Issue 3 2008Priya Rajavelu M.Sc. Abstract In this study, we compared several immune parameters in tuberculosis (TB) and nontuberculosis (NTB) pleurisy to gain an understanding of the mechanism behind enhanced Th1 apoptosis that occurs at sites of active Myobacterium tuberculosis (M. tuberculosis) infection. An initial evaluation of the accumulated cytokines in pleural fluid (PF) demonstrated that both TB and NTB pleurisy were associated with prointflammatory cytokines, while only TB pleurisy had augmented expression of interferon (IFN)-, and soluble Fas ligand (sFASL). Despite enhanced expression of the apoptosis-inducing molecule in TB pleurisy, T cells derived from both types of pleurisy exhibited significant apoptosis. In both groups, T-cell apoptosis correlated with low expression of CD80 on PF-derived macrophages and elevated accumulation of TGF-, in the PF. A causative correlation between TGF-, and low CD80 expression in the two groups was established by in vitro studies demonstrating TGF-, inhibition of CD80 upregulation in a macrophage cell line. Together, the findings allude to the possibility that activation in the absence of appropriate CD80 costimulation is the mechanism that leads to T-cell apoptosis at sites of active M. tuberculosis infection. Furthermore, the findings also indicate that T-cell apoptosis is perhaps a host regulatory mechanism to limit inflammation, rather than a pathogen-induced immune deviation. [source] Pathogenesis of Helicobacter pylori InfectionHELICOBACTER, Issue 2004Paul Hofman ABSTRACT Research in the last year has provided new insights into the function of the the cag -associated type IV secretion system and the vacuolating toxin VacA. A quite new aspect was disclosed by the finding that Helicobacter pylori in Mongolian gerbils colonizes a very distinct topology in the gastric mucous layer, obviously providing optimal conditions for long-term survival. Further research activities focused on H. pylori ammonia and metal metabolism as well as on bacterial stress defence mechanisms. Differential expression of approximately 7% of the bacterial genome was found at low pH suggesting that H. pylori has evolved a multitude of acid-adaptive mechanisms. VacA was shown to interrupt phagosome maturation in macrophage cell lines as well as to modulate and interfere with T lymphocyte immunological functions. Gastric mucosa as well as the H. pylori -infected epithelial cell line AGS strongly express IL-8 receptor A and B, which might contribute to the augmentation of the inflammatory response. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. The chronic imbalance between apoptosis and cell proliferation is the first step of gastric carcinogenesis. In this regard, it was demonstrated that coexpression of two H. pylori proteins, CagA and HspB, in AGS cells, caused an increase in E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein. Taken together, we now have a better understanding of the role of different virulence factors of H. pylori. There is still a lot to be learned, but the promising discoveries summarized here, demonstrate that the investigation of the bacterial survival strategies will give novel insights into pathogenesis and disease development. [source] Activated ,2 macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-,B activation in macrophage-derived cell linesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010Leandro C. Cáceres Abstract Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP-9) expression and protein secretion through the activation of MAPK-ERK and NF-,B signaling pathways. Previously, we demonstrated that activated ,2 -macroglulin (,2M*) through the interaction with its receptor low-density lipoprotein receptor-related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK-ERK1/2. In the present work, we examined whether ,2M*/LRP1interaction could induce the MMP-9 production in J774 and Raw264.7 macrophage-derived cell lines. It was shown that ,2M* promoted MMP-9 expression and protein secretion by LRP1 in both macrophage-derived cell lines, which was mediated by the activation of MAPK-ERK1/2 and NF-,B. Both intracellular signaling pathways activated by ,2M* were effectively blocked by calphostin-C, suggesting involvement of PKC. In addition, we demonstrate that ,2M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA-AM, the ,2M*-induced MAPK-ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF-,B, it was shown that the ,2M*-induced MMP-9 protein secretion was inhibited, indicating that the MMP production promoted by the ,2M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607,617, 2010. © 2010 Wiley-Liss, Inc. [source] Lycopene Inhibits LPS-Induced Proinflammatory Mediator Inducible Nitric Oxide Synthase in Mouse Macrophage CellsJOURNAL OF FOOD SCIENCE, Issue 1 2007Mohamed M. Rafi ABSTRACT:, Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 ,M) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. [source] A nitric oxide (NO)-releasing derivative of gabapentin, NCX 8001, alleviates neuropathic pain-like behavior after spinal cord and peripheral nerve injuryBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2004Wei-Ping Wu Nitric oxide (NO) participates, at least in part, to the establishment and maintenance of pain after nerve injury. Therefore, drugs that target the NO/cGMP signaling pathway are of interest for the treatment of human neuropathic pain. Various compounds endowed with NO-releasing properties modulate the expression and function of inducible nitric oxide synthase (iNOS), the key enzyme responsible for sustained NO production under pathological conditions including neuropathic pain. With this background, we synthesized a new chemical entity, [1-(aminomethyl)cyclohexane acetic acid 3-(nitroxymethyl)phenyl ester] NCX8001, which has a NO-releasing moiety bound to gabapentin, a drug currently used for the clinical management of neuropathic pain. We examined the pharmacological profile of this drug with respect to its NO-releasing properties in vitro as well as to its efficacy in treating neuropathic pain conditions (allodynia) consequent to experimental sciatic nerve or spinal cord injuries. NCX8001 (1,30 ,M) released physiologically relevant concentrations of NO as it induced a concentration-dependent activation of soluble guanylyl cyclase (EC50=5.6 ,M) and produced consistent vasorelaxant effects in noradrenaline-precontracted rabbit aortic rings (IC50=1.4 ,M). NCX8001, but not gabapentin, counteracted in a concentration-dependent fashion lipopolysaccharide-induced overexpression and function of iNOS in RAW264.7 macrophages cell line. Furthermore, NCX8001 also inhibited the release of tumor necrosis factor alpha (TNF,) from stimulated RAW264.7 cells. NCX8001 (28,280 ,mol kg,1, i.p.) reduced the allodynic responses of spinal cord injured rats in a dose-dependent fashion while lacking sedative or motor effects. In contrast, gabapentin (170,580 ,mol kg,1, i.p.) resulted less effective and elicited marked side effects. NCX8001 alleviated the allodynia-like responses of rats to innocuous mechanical or cold stimulation following lesion of the sciatic nerve. This effect was not shared by equimolar doses of gabapentin. Potentially due to the slow releasing kinetics of NO, NCX8001 alleviated pain-like behaviors in two rat models of neuropathic pain in a fashion that is superior to its parent counterpart gabapentin. This new gabapentin derivative, whose mechanism deserves to be explored further, offers new hopes to the treatment of human neuropathic pain. British Journal of Pharmacology (2004) 141, 65,74. doi:10.1038/sj.bjp.0705596 [source] L-selectin and E-selectin expressed on monocytes mediating Ehrlichia chaffeensis attachment onto host cellsFEMS MICROBIOLOGY LETTERS, Issue 2 2003Jian-zhi Zhang Abstract Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is an obligatory intracellular bacterium that exhibits monocytic host cell tropism. Ehrlichiae must enter the host cell, and then establish infection. The tropism of E. chaffeensis for monocytes suggests that the cell contains some specific surface components that mediate E. chaffeensis attachment and entry into host cells. In this study, host cell surface components that play a role in ehrlichial attachment were identified using a human monocyte/macrophage cell line, THP-1. E. chaffeensis attachment to THP-1 cells was partially blocked in the presence of antibodies to E-selectin and L-selectin, but not by antibodies to P-selectin, integrin ,m, integrin ,x, or normal mouse IgG as determined by real time polymerase chain reaction. Conversely, in HeLa cells that do not exhibit surface expression of E-selectin and L-selectin, antibodies to these cell surface proteins did not inhibit E. chaffeensis attachment. These findings indicate that E-selectin and L-selectin are cell surface proteins that might act as co-receptors and contribute to E. chaffeensis attachment and entry into THP-1. [source] | ||||||||||||||||