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Trivedi, Mahendra Kumar, Alice Branton, Dahryn Trivedi, Gopal Nayak, Sambhu Charan Mondal, and Snehasis Jana. "Immunomodulatory Effect of the Biofield Energy Treated Cell Growth Medium (DMEM) and FBS in Immune Cells (Murine Macrophage RAW 264.7)." Journal of Cellular Immunology and Serum Biology 3, no. 2 (2018): 1–5. https://doi.org/10.15436/2471-5891.18.1946.
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The objective of the present study was to evaluate the immunomodulatory effect of Biofield Energy Treatment (The Trivedi Effect®) on Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) in murine macrophage cells (RAW 264.7). The study parameters were evaluated using cell viability by MTT assay and estimation of proinflammatory cytokine like tumor necrosis factor - alpha (TNF-α) on immunomodulation using enzyme linked immune sorbent assay (ELISA). The cell viability using MTT assay data showed that more than 80% cell viability was observed in all the tested groups compared to the baseline control group (G1). The characteristics of cell morphology in the Biofield Energy Treated DMEM without FBS (G6) group showed some changes in the cell morphology as evidenced a transition from spherical to elongate and also showed maturation compared to the G1 group. The level of TNF-α expression was significantly reduced by 23.15% in the G3 group compared to the G1 group. Moreover, the secretion of TNF-α was altered by 38.06%, 35.77%, and 282.49% in the G4, G5, and G6 groups, respectively compared to the G1 group. The overall results demonstrated that the Trivedi Effect® - Consciousness Energy Healing Treatment has an impact on DMEM and FBS by alteration of cell morphology and reducing the level of TNF-α expression in immune cell (RAW 264.7). Therefore, the Trivedi Effect® treated DMEM and FBS might be useful as an immunomodulator for various immune-related disorders like Graves’ disease, rheumatoid arthritis, multiple sclerosis, etc. <strong>Source:</strong> https://www.trivedieffect.com/science/immunomodulatory-effect-of-the-biofield-energy-treated-cell-growth-medium-dmem-and-fbs-in-immune-cells-murine-macrophage-raw-264-7/ https://www.ommegaonline.org/article-details/Immunomodulatory-Effect-of-the-Biofield-Energy-Treated-Cell-Growth-Medium-DMEM-and-FBS-in-Immune-Cells-Murine-Macrophage-RAW-2647/1968
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Tellez, Angela, Milena Corredig, Lubov Y. Brovko, and Mansel W. Griffiths. "Characterization of immune-active peptides obtained from milk fermented byLactobacillus helveticus." Journal of Dairy Research 77, no. 2 (2010): 129–36. http://dx.doi.org/10.1017/s002202990999046x.
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The objectives of this research were to confirm the effect of compounds derived from milk fermented byLactobacillus helveticus(LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were testedin vitrofor immuno-modulating activity using murine macrophages (RAW 264·7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), and Interleukin-1β (IL1-β)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from β-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-α showed a direct correlation between protein concentration and TNF-α release. The fraction contained four novel peptides, three derived from the hydrolysis of β-casein and one from the hydrolysis of α-lactalbumin. These results confirm that fermentation of milk byLactobacillus helveticus(LH-2) results in the production of specific peptides capable of modulating macrophage activity.
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Jofre-Monseny, Laia, Sonia de Pascual-Teresa, Eva Plonka, et al. "Differential effects of apolipoprotein E3 and E4 on markers of oxidative status in macrophages." British Journal of Nutrition 97, no. 5 (2007): 864–71. http://dx.doi.org/10.1017/s0007114507669219.
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ApoE is secreted by macrophages at the lesion site of the atherosclerotic plaque, where it is thought to play a protective role against atherosclerosis independently of its effects on lipid metabolism. Of the three common isoforms for apoE, apoE4 is associated with higher risk of cardiovascular disease (CVD). In vitro studies have shown that recombinant apoE may act as an antioxidant in an isoform-dependent manner (E2>E3>E4). The oxidative status of the macrophages plays a key role in the process of atherosclerosis. In the present study the possible differential actions of apoE3 and apoE4 on several parameters of oxidative status were determined in stably transfected murine macrophages (RAW 264·7-apoE3 and -apoE4). No differences between genotypes were observed after peroxide challenge in either protection against cytotoxicity or in cell membrane oxidation, and modest differences were observed in the non-enzymatic antioxidants (glutathione and α-tocopherol) in apoE3 v. apoE4 macrophages. Importantly, cells secreting apoE4 showed increased membrane oxidation under basal conditions, and produced more NO and superoxide anion radicals than the apoE3 macrophages after stimulation. The present data suggest that apoE genotype influences the oxidative status of macrophages, and this could partly contribute to the higher CVD risk observed in apoE4 carriers.
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Tabouret, G., I. Vouldoukis, C. Duranton, et al. "Oestrus ovis (Diptera: Oestridae): effects of larval excretory/secretory products on nitric oxide production by murine RAW 264·7 macrophages." Parasite Immunology 23, no. 3 (2001): 111–19. http://dx.doi.org/10.1046/j.1365-3024.2001.00355.x.
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Chon, H., B. Choi, E. Lee, S. Lee, and G. Jeong. "Immunomodulatory effects of specific bacterial components ofLactobacillus plantarumKFCC11389P on the murine macrophage cell line RAW 264·7." Journal of Applied Microbiology 107, no. 5 (2009): 1588–97. http://dx.doi.org/10.1111/j.1365-2672.2009.04343.x.
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Koc, A., T. Ozkan, A. Z. Karabay, A. Sunguroglu, and F. Aktan. "Effect of L-carnitine on the synthesis of nitric oxide in RAW 264·7 murine macrophage cell line." Cell Biochemistry and Function 29, no. 8 (2011): 679–85. http://dx.doi.org/10.1002/cbf.1807.
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Keen, Imelda, Traian V. Chirila, Zeke Barnard, Z. Zainuddin, and Andrew K. Whittaker. "Degradable Hydrogels for Tissue Engineering - Part II: Responses of Fibroblasts and Macrophages to Linear PHEMA." Journal of Biomimetics, Biomaterials and Tissue Engineering 8 (November 2010): 91–104. http://dx.doi.org/10.4028/www.scientific.net/jbbte.8.91.
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A series of linear poly(2-hydroxyethyl methacrylate) (PHEMA) with defined molecular weights (MW) and narrow molecular distributions were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using cumyl dithiobenzoate (CDB) as a chain transfer agent. Murine fibroblasts (3T3) were exposed to eluates from various PHEMA samples, washed or unwashed, and with or without dithioester end groups. After 72 hrs in cell culture, no cytotoxic response was elicited by the polymer samples devoid of dithioester end groups, and which also underwent a thorough washing regime. Specimens throughout the entire MW range were internalized by a macrophage (cell line Raw 264), suggesting that such polymers can be used as models for studying the biodegradation of PHEMA.
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Tjandrawinata, R. R., L. Hawel, and C. V. Byus. "Regulation of putrescine export in lipopolysaccharide or IFN-gamma-activated murine monocytic-leukemic RAW 264 cells." Journal of Immunology 152, no. 6 (1994): 3039–52. http://dx.doi.org/10.4049/jimmunol.152.6.3039.
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Abstract The regulation of putrescine/polyamine export out of the cell was investigated during activation of monocytic-leukemic RAW 264 cells with LPS and IFN-gamma. The RAW 264 cells exported putrescine constitutively at a significant rate into the culture medium. This export process appeared to be selective for putrescine in that only a small amount of other polyamines (spermidine and N1-acetylspermidine) was found in the culture medium. LPS and IFN-gamma alone and in combination markedly stimulated putrescine export and nitrite production throughout a 24-h period. The efflux of putrescine but not nitrite was further increased by the addition of ornithine (the amino acid precursor of putrescine) to the culture medium. LPS and ornithine also stimulated the intracellular accumulation of putrescine in primary inflammatory macrophages and the export of putrescine into the peritoneal exudate of the mouse. A detailed comparison of the steady state rates of accumulation of intracellular putrescine/polyamines and the rate of putrescine efflux from the cells constitutively and after LPS, IFN-gamma, and ornithine indicated that a surprisingly large fraction of total polyamine biosynthesis is comprised of exported putrescine. The observed dose-dependent inhibition of putrescine export with the drug verapamil implicated the involvement of a specific membrane transport system sensitive to calcium influx in this process. The data are discussed in regard to the potential involvement of putrescine export in the regulation of intracellular polyamine levels, cell differentiation, and macrophage-mediated cytotoxicity.
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Homsani, Fortune, Gleyce Moreno, Camila Siqueira, Juliana Grechi, André Luis Santos, and Carla Holandino. "Cellular Alterations Induced by Candida albicans RC Nosodes: an in vitro Study." International Journal of High Dilution Research - ISSN 1982-6206 11, no. 40 (2021): 209–10. http://dx.doi.org/10.51910/ijhdr.v11i40.600.
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Introduction: Candidiasis is an opportunist infection, caused by yeast of the genus Candida, which emerges as one of the main causes of systemic infections in hospitalized patients. Candida albicans is the most common causing agent of these infections. According to the Brazilian Homeopathic Pharmacopeia[1], nosodes are medicines compounded from chemically undefined biological products. Living nosodes are prepared using the etiologic agent of an illness in its infective form, were first developed by Brazilian physician Roberto Costa (RC). Roberto Costa’s research indicated that living nosodes present a higher capability to stimulate the host’s immunological system [2]. 
 Aim: This study aims to evaluate cellular alterations induced in C. albicans yeasts and RAW 264-7 macrophages by Candida albicans RC. 
 Methodology: To prepare Candida albicans RC, one part of C. albicans infective yeast suspension (108 cell/ml) was diluted in 9 parts of sterile distilled water and submitted to 100 mechanical succussions. This process was successively repeated to the potencies of 12x and 30x1. Water 30x was prepared by the same technique, as control. The cell viability of C. albicans previously treated with nosodes in both potencies and respective controls was evaluated using the samples at the concentration of 10% (V/V), in a volume of 1ml, distributed in 1-3 days. The viability of the yeast cells was analyzed by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolic) (5mg/ml) assay [3] and by Propidium Iodide (PI) incorporation methods. Additionally, using macrophages RAW 264-7 as a cell model, Nitric Oxide (NO) production and cell viability were also evaluated. For this, the following protocol of cell treatment was employed: on each experimental day, RAW 264-7 cells were treated 4 times (4 stimuli) with RC nosode 30x at the concentration of 10% (V/V). 
 Results: The nosodes (12x and 30x) did not present cytotoxic effects on macrophage cells (n=1), or on C. albicans yeasts (n=2), as detected by MTT and PI methods. Moreover, no statistically significant differences on NO production were detected among the experimental groups (n=6). 
 Conclusion: Preliminary results of in vitro assays indicate that nosodes (12x and 30x) do not alter mitochondrial activity or cell viability of C. albicans. Similarly, treatment by RC nosodes does not seem to alter NO release and mitochondrial activity of RAW macrophages. New experiments are being performed to confirm these preliminary data.
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Rao, P., L. A. Falk, S. F. Dougherty, T. Sawada, and D. H. Pluznik. "Colchicine down-regulates lipopolysaccharide-induced granulocyte-macrophage colony-stimulating factor production in murine macrophages." Journal of Immunology 159, no. 7 (1997): 3531–39. http://dx.doi.org/10.4049/jimmunol.159.7.3531.
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Abstract Activation of macrophages by LPS and taxol results in production of IL-1, IL-6, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF), which are involved in regulating hemopoiesis, inflammation, and immune responses. Microtubules are proposed as a target site for LPS interaction(s), based on similarities between the effects of the tubulin-binding drug taxol and LPS. To clarify the role of microtubules in LPS-induced GM-CSF expression in macrophages, we examined whether microtubule depolymerizing agents affect GM-CSF production in macrophages. Pretreatment with colchicine impaired LPS induction of GM-CSF in RAW 264 cells, and studies using stable transfectants revealed that colchicine impaired the transcriptional responsiveness of a reporter gene driven by a GM-CSF promoter sequence. Colchicine inhibition of the GM-CSF response correlated with decreases in the mRNA levels of beta-tubulin; maximal inhibition of both events was observed 4 h after addition of colchicine. Microtubule agents inhibited LPS induction of IL-6 and TNF-alpha, while the induction of both IL-1beta and inducible nitric oxide synthase was unaltered, suggesting that LPS activates microtubule-dependent and -independent pathways. Interestingly, LPS stimulation of macrophages down-regulated levels of beta-tubulin transcripts, implying that LPS interacts with an element(s) of the microtubule network in vivo, activating pathways regulating transcription of beta-tubulin. The ability of both colchicine and LPS to modulate transcription of beta-tubulin suggests that this event does not per se underlie the inhibitory effect of colchicine on LPS-induced GM-CSF expression. These data led us to conclude that colchicine inhibits LPS induction of GM-CSF by affecting microtubule-dependent costimulatory signaling pathways that synergize with primary LPS-triggered responses.
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Pool, Edmund John. "The effects of silver nanoparticles on RAW 264 7 Macrophages and human whole blood cell cultures." Frontiers in Bioscience 24, no. 2 (2019): 347–65. http://dx.doi.org/10.2741/4722.
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Makkonen, Niina, Antero Salminen, Michael J. Rogers, et al. "Contrasting effects of alendronate and clodronate on RAW 264 macrophages: the role of a bisphosphonate metabolite." European Journal of Pharmaceutical Sciences 8, no. 2 (1999): 109–18. http://dx.doi.org/10.1016/s0928-0987(98)00065-7.
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Tebo, J. M., C. Wu, Y. Ohmori та T. A. Hamilton. "Murine IκBα negatively regulates κB-dependent transcription in LPS-stimulated RAW 264.7 macrophages". Cytokine 6, № 5 (1994): 563. http://dx.doi.org/10.1016/1043-4666(94)90226-7.
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Shao, Feng, Layla Panahipour, Mariane Beatriz Sordi, Fangrui Tang, Ronghua Liu, and Reinhard Gruber. "Heartwood of Dalbergia cochinchinensis: 4,7,2′-Trihydroxy-4′-methoxyisoflavanol and 6,4′-Dihydroxy-7-methoxyflavane Reduce Cytokine and Chemokine Expression In Vitro." Molecules 27, no. 4 (2022): 1321. http://dx.doi.org/10.3390/molecules27041321.
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Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2′-trihydroxy-4′-methoxyisoflavanol (472T4MIF) and 6,4′-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1β and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells.
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Miranda, Diego Garcia, Lucas de Paula Ramos, Nicole Fernanda dos Santos Lopes, et al. "Ketoprofen Associated with Hyaluronic Acid Hydrogel for Temporomandibular Disorder Treatment: An In Vitro Study." Gels 10, no. 12 (2024): 811. https://doi.org/10.3390/gels10120811.
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Temporomandibular disorders (TMD) are a public health problem that affects around 12% of the global population. The treatment is based on analgesics, non-steroidal anti-inflammatory, corticosteroids, anticonvulsants, or arthrocentesis associated with hyaluronic acid-based viscosupplementation. However, the use of hyaluronic acid alone in viscosupplementation does not seem to be enough to regulate the intra-articular inflammatory process. So, we propose to develop and evaluate the physicochemical and biological properties in vitro of hyaluronic acid hydrogels (HA) associated with ketoprofen (KET) as a new therapeutic treatment for TMD. The hydrogels were synthesized with 3% HA and 0.125, 0.250, 0.500, or 1% KET. Physicochemical analyses of Attenuated Total reflectance-Fourier transform infrared spectroscopy (FTIR), Thermogravimetry (TGA), Rheology by Frequency, Amplitude sweeps, temperature ramp, and scanning electron microscopy (SEM) were performed with or without sterilization and cycled. Cytocompatibility and genotoxicity (micronucleus assay) were performed in mouse macrophages (RAW 264-7) for 24 h. Results: FTIR spectrum showed characteristic absorptions of HA and KET. In the TGA, two mass loss peaks were observed, the first representing the water evaporation at 30 and 100 °C, and the second peaks between 200 and 300 °C, indicating the degradation of HA and KET. Rheology tests in the oscillatory regime classified the hydrogels as non-Newtonian fluids, time-dependent, and thixotropic. Mouse macrophages (RAW 264-7) presented viability of 83.6% for HA, 50.7% for KET, and 92.4%, 66.1%, 65.3%, and 87.7% for hydrogels, in addition to the absence of genotoxicity. Conclusions: Hyaluronic acid associated with ketoprofen shows satisfactory physicochemical and biological properties for use as viscosupplementation. As a limiting point of this study, further research is needed to evaluate the pharmacodynamic, toxicological, and pharmacokinetic characteristics of a complete organism
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Uchiyama, Ryosuke, Ikuo Kawamura, Takao Fujimura, et al. "Involvement of Caspase-9 in the Inhibition of Necrosis of RAW 264 Cells Infected with Mycobacterium tuberculosis." Infection and Immunity 75, no. 6 (2007): 2894–902. http://dx.doi.org/10.1128/iai.01639-06.
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ABSTRACT In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.
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Gorecka-Tisera, Alicja M., K. W. Snowdowne, and A. B. Borle. "Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing." Cellular Immunology 100, no. 2 (1986): 411–21. http://dx.doi.org/10.1016/0008-8749(86)90040-7.
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Siqueira, Camila, Rafaela De Mendonça, Venício Da Veiga, et al. "Biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2) and inactivated influenza A (H3N2) virus at 12x and 30x in MDCK cells and RAW-264-7 macrophages." International Journal of High Dilution Research - ISSN 1982-6206 11, no. 40 (2021): 180–81. http://dx.doi.org/10.51910/ijhdr.v11i40.597.
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Background: "Roberto Costa’s Biotherapics" are homeopathic remedies prepared from intact microorganisms which have been proposed for treatment of diseases like influenza. 
 Aim: This study aimed to compare the biochemical effects, in MDCK cells and RAW-264-7 macrophages, of biotherapics prepared from intact influenza virus diluted in water as well as from a sample of the same virus inactivated by ethanol 70% (v / v), both in the homeopathic potencies of 12x and 30x. Water 30x, non-dynamized water and cells without treatment (control cells) were used as control.
 Methodology: Treatments were performed by incubating MDCK cells with DMEM medium added in a 1:10 ratio for 6 times (3 different aliquots per day) or 18 times (up to 4 aliquots per day) in each experimental situation. Each aliquot was added with an interval of at least 2 hours. After that, the mitochondrial activity of MDCK cells was analyzed by MTT assay. The effects of treatments with intact biotherapics on MDCK cells respiratory parameters were studied using high resolution respirometry (Oroboros Oxygraph-O2K). RAW-264-7 macrophages were treated with intact and inactivated biotherapic 30x (4 treatments, 24 hours) to verify the nitric oxide production. These macrophages were also submitted to MTT assay.
 Results: Both biotherapic preparations 1x (intact and inactivated virus sample) were analyzed by transmission electronic microscopy. The presence of virus particles was detected when water was used as solvent. The use of ethanol as biotherapic solvent induced complete virus lysis. There was no alteration in cell osmolarity revealed by neutral red assay, when 10% of each test solution was used. Cellular viability analyzed by MTT method increased when MDCK cells were treated with 18 stimuli of inactivated biotherapic 30x when compared to intact biotherapic 30x (p0.05) were detected when these cells were compared to control cells. 
 The maximum respiratory capacity of MDCK cells increased after treatment with 18 stimuli of intact biotherapic 30x when compared to control cells. However, no statistically significant differences (p>0.05) induced by biotherapics in macrophage cells were observed by MTT and nitric oxide assays. Moreover, a reduction in nitric oxide was observed in macrophages treated with dynamized water when compared to control cells. 
 Conclusions: These results indicate that the method of biotherapic compounding (intact or inactivated virus as starting point) can modify the cellular parameters with the tendency to increase cellular response with longer treatments and higher potencies.
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Shimoyama, Tadashi, Yoshiaki Okano, Yukiteru Fujishima, et al. "Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia." Blood 124, no. 21 (2014): 1456. http://dx.doi.org/10.1182/blood.v124.21.1456.1456.
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Abstract Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments. Methods, Results and Discussion: In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis. In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo. Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.
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Kwak, Chungshil, Hye In Choi, and Jiwon Yang. "Antioxidant activity of Rosa multiflora Thunb. flower extract and suppressive activity on proinflammatory mediator production in lipopolysaccharide- stimulated RAW 264.7 macrophages." Functional Foods in Health and Disease 6, no. 5 (2016): 265. http://dx.doi.org/10.31989/ffhd.v6i5.248.
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Background: Oxidative stress and inflammation are associated with various ageing-related chronic diseases. The fruits and roots of Rosa multiflora Thunb. have been used in medicine for the treatment of edema and inflammatory diseases in Eastern Asia. Dried Rosa multiflora Thunb. flower (RMF) have been consumed as a tea in Korea, but reports on the biological activity of RMF are lacking. We evaluated the in vitro antioxidant and anti-inflammatory effects of an ethanol extract from RMF as well as various solvent fractions from the extract.Methods: The ethanol extract (Et) of RMP was fractionated sequentially by hexane (Hx), dichloromethane (DM), ethylacetate (EA), n-butanol (Bt) and distilled water (DW). Total phenolic content and total flavonoid content, scavenging activities of the 2,2-diphenyl-1 picrylhydrazyl radical and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical and ferric-reducing antioxidant power were measured. Anti-inflammatory effects in terms of levels of nitric oxide and prostaglandin (PG) E2 and production of proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages were measured, and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were measured by Western blot analysis.Results: EA treatment showed the highest total phenolic and total flavonoid content and strongest antioxidant activity, followed by Bt and Et, measured by three different methods. Treatment with Et and all fractions significantly suppressed (p<0.05) nitric oxide production in a dose-dependent manner in LPS-treated RAW 264.7 macrophages via reduction of expression of iNOS protein. Treatment with Et, DM and EA significantly suppressed (p<0.05) PGE2 production induced by LPS treatment, however, only Bt treatment significantly reduced (p<0.05) the expression of COX-2 protein. Treatment with DM, EA and Bt suppressed IL-6 production significantly (p<0.05) in LPS-treated RAW 264.7 macrophages, and treatment with Et, DM, EA and Bt suppressed TNF-α production significantly (p<0.05).Conclusions: These data suggest that the ethanol extract of Rosa multiflora Thunb. flower and its dichloromethan, ethylacetate and n-butanol fractions have potent antioxidant and/or anti-inflammatory activities.Keywords: Rosa multiflora Thunb. flower, antioxidant activity, anti-inflammatory activity, RAW 264 7 macrophages, cytokines, iNOS, COX-2
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Cho, Young-Chang, Jiyoung Park, and Sayeon Cho. "Anti-Inflammatory and Anti-Oxidative Effects of luteolin-7-O-glucuronide in LPS-Stimulated Murine Macrophages through TAK1 Inhibition and Nrf2 Activation." International Journal of Molecular Sciences 21, no. 6 (2020): 2007. http://dx.doi.org/10.3390/ijms21062007.
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Various herbal extracts containing luteolin-7-O-glucuronide (L7Gn) have been traditionally used to treat inflammatory diseases. However, systemic studies aimed at elucidating the anti-inflammatory and anti-oxidative mechanisms of L7Gn in macrophages are insufficient. Herein, the anti-inflammatory and anti-oxidative effects of L7Gn and their underlying mechanisms of action in macrophages were explored. L7Gn inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages by transcriptional regulation of inducible NO synthase (iNOS) in a dose-dependent manner. The mRNA expression of inflammatory mediators, including cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α), was inhibited by L7Gn treatment. This suppression was mediated through transforming growth factor beta-activated kinase 1 (TAK1) inhibition that leads to reduced activation of nuclear factor-κB (NF-κB), p38, and c-Jun N-terminal kinase (JNK). L7Gn also enhanced the radical scavenging effect and increased the expression of anti-oxidative regulators, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone oxidoreductase 1 (NQO1), by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activation. These results indicate that L7Gn exhibits anti-inflammatory and anti-oxidative properties in LPS-stimulated murine macrophages, suggesting that L7Gn may be a suitable candidate to treat severe inflammation and oxidative stress.
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Lu, Jing, Joshua Reese, Ying Zhou, and Emmet Hirsch. "Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophage cells." Journal of Endocrinology 224, no. 2 (2014): 183–94. http://dx.doi.org/10.1530/joe-14-0470.
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Parturition is an inflammatory process mediated to a significant extent by macrophages. Progesterone (P4) maintains uterine quiescence in pregnancy, and a proposed functional withdrawal of P4 classically regulated by nuclear progesterone receptors (nPRs) leads to labor. P4 can affect the functions of macrophages despite the reported lack of expression of nPRs in these immune cells. Therefore, in this study we investigated the effects of the activation of the putative membrane-associated PR on the function of macrophages (a key cell for parturition) and discuss the implications of these findings for pregnancy and parturition. In murine macrophage cells (RAW 264.7), activation of mPRs by P4 modified to be active only extracellularly by conjugation to BSA (P4BSA, 1.0×10−7 mol/l) caused a pro-inflammatory shift in the mRNA expression profile, with significant upregulation of the expression of cyclooxygenase 2 (COX2 (Ptgs2)), Il1B, and Tnf and downregulation of membrane progesterone receptor alpha (Paqr7) and oxytocin receptor (Oxtr). Pretreatment with PD98059, a MEK1/2 inhibitor, significantly reduced P4BSA-induced expression of mRNA of Il1B, Tnf, and Ptgs2. Inhibition of protein kinase A (PKA) by H89 blocked P4BSA-induced expression of Il1B and Tnf mRNA. P4BSA induced rapid phosphorylation of MEK1/2 and CREB (a downstream target of PKA). This phosphorylation was inhibited by pretreatment with PD98059 and H89, respectively, revealing that MEK1/2 and PKA are two of the components involved in mPR signaling. Taken together, these results indicate that changes in membrane progesterone receptor alpha expression and signaling in macrophages are associated with the inflammatory responses; and that these changes might contribute to the functional withdrawal of P4 related to labor.
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Tanaka, Miori, Akari Sato, Yoshimi Kishimoto, Hideaki Mabashi-Asazuma, Kazuo Kondo, and Kaoruko Iida. "Gallic Acid Inhibits Lipid Accumulation via AMPK Pathway and Suppresses Apoptosis and Macrophage-Mediated Inflammation in Hepatocytes." Nutrients 12, no. 5 (2020): 1479. http://dx.doi.org/10.3390/nu12051479.
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Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease, sometimes ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). Various hits including excessive hepatic steatosis, oxidative stress, apoptosis, and inflammation, contribute to NASH development. Gallic acid (GA), a natural polyphenol, was reported to exert a protective effect on hepatic steatosis in animal models, but the precise molecular mechanisms remain unclear. Here, we examined the effect of GA on hepatic lipid accumulation, apoptosis, and inflammatory response caused by hepatocyte–macrophage crosstalk. We demonstrated that GA attenuated palmitic acid (PA)-induced fat accumulation via the activation of AMP-activated protein kinase (AMPK) in HepG2 cells. GA also ameliorated cell viability and suppressed apoptosis-related gene expression and caspase 3/7 activity induced by PA and H2O2. In a co-culture of lipid-laden Hepa 1-6 hepatocytes and RAW 264 macrophages, GA reduced inflammatory mediator expression and induced antioxidant enzyme expression. These results indicate that GA suppresses hepatic lipid accumulation, apoptosis, and inflammation caused by the interaction between hepatocytes and macrophages. The potential effects of GA observed in our study could be effective in preventing NASH and its complications.
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Hon, Wei-Min, Shabbir Moochhala, and Hoon-Eng Khoo. "Adenosine and its receptor agonists potentiate nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages." Life Sciences 60, no. 16 (1997): 1327–35. http://dx.doi.org/10.1016/s0024-3205(97)00078-7.
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Brahmi, Fatiha, Thomas Nury, Meryam Debbabi, et al. "Evaluation of Antioxidant, Anti-Inflammatory and Cytoprotective Properties of Ethanolic Mint Extracts from Algeria on 7-Ketocholesterol-Treated Murine RAW 264.7 Macrophages." Antioxidants 7, no. 12 (2018): 184. http://dx.doi.org/10.3390/antiox7120184.
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The present study consisted in evaluating the antioxidant, anti-inflammatory and cytoprotective properties of ethanolic extracts from three mint species (Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L.) Huds (MR)) with biochemical methods on murine RAW 264.7 macrophages (a transformed macrophage cell line isolated from ascites of BALB/c mice infected by the Abelson leukemia virus). The total phenolic, flavonoid and carotenoid contents were determined with spectrophotometric methods. The antioxidant activities were quantified with the Kit Radicaux Libres (KRLTM), the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. The MS extract showed the highest total phenolic content, and the highest antioxidant capacity, while the MR extract showed the lowest total phenolic content and the lowest antioxidant capacity. The cytoprotective and anti-inflammatory activities of the extracts were quantified on murine RAW 264.7 macrophages treated with 7-ketocholesterol (7KC; 20 µg/mL: 50 µM) associated or not for 24 h and 48 h with ethanolic mint extracts used at different concentrations (25, 50, 100, 200 and 400 µg/mL). Under treatment with 7KC, an important inhibition of cell growth was revealed with the crystal violet test. This side effect was strongly attenuated in a dose dependent manner with the different ethanolic mint extracts, mainly at 48 h. The most important cytoprotective effect was observed with the MS extract. In addition, the effects of ethanolic mint extracts on cytokine secretion (Interleukin (IL)-6, IL-10, Monocyte Chemoattractant Protein (MCP)-1, Interferon (IFN)-ϒ, Tumor necrosis factor (TNF)-α) were determined at 24 h on lipopolysaccharide (LPS, 0.2 µg/mL)-, 7KC (20 µg/mL)- and (7KC + LPS)-treated RAW 264.7 cells. Complex effects of mint extracts were observed on cytokine secretion. However, comparatively to LPS-treated cells, all the extracts strongly reduce IL-6 secretion and two of them (MP and MR) also decrease MCP-1 and TNF-α secretion. However, no anti-inflammatory effects were observed on 7KC- and (7KC + LPS)-treated cells. Altogether, these data bring new evidences on the potential benefits (especially antioxidant and cytoprotective properties) of Algerian mint on human health.
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Gottstein, Nicole, Benjamin A. Ewins, Clair Eccleston, et al. "Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function." British Journal of Nutrition 89, no. 5 (2003): 607–15. http://dx.doi.org/10.1079/bjn2003820.
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There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the presentin vitrostudies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264·7) activated with interferon γ plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor α (TNF-α) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-α in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the currentin vitrostudies are relevant to the aetiology of coronary artery diseasein vivo.
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Shvedova, Anna A., Elena R. Kisin, Robert Mercer, et al. "Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 5 (2005): L698—L708. http://dx.doi.org/10.1152/ajplung.00084.2005.
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Single-walled carbon nanotubes (SWCNT) are new materials of emerging technological importance. As SWCNT are introduced into the life cycle of commercial products, their effects on human health and environment should be addressed. We demonstrated that pharyngeal aspiration of SWCNT elicited unusual pulmonary effects in C57BL/6 mice that combined a robust but acute inflammation with early onset yet progressive fibrosis and granulomas. A dose-dependent increase in the protein, LDH, and γ-glutamyl transferase activities in bronchoalveolar lavage were found along with accumulation of 4-hydroxynonenal (oxidative biomarker) and depletion of glutathione in lungs. An early neutrophils accumulation ( day 1), followed by lymphocyte ( day 3) and macrophage ( day 7) influx, was accompanied by early elevation of proinflammatory cytokines (TNF-α, IL-1β; day 1) followed by fibrogenic transforming growth factor (TGF)-β1 (peaked on day 7). A rapid progressive fibrosis found in mice exhibited two distinct morphologies: 1) SWCNT-induced granulomas mainly associated with hypertrophied epithelial cells surrounding SWCNT aggregates and 2) diffuse interstitial fibrosis and alveolar wall thickening likely associated with dispersed SWCNT. In vitro exposure of murine RAW 264.7 macrophages to SWCNT triggered TGF-β1 production similarly to zymosan but generated less TNF-α and IL-1β. SWCNT did not cause superoxide or NO·production, active SWCNT engulfment, or apoptosis in RAW 264.7 macrophages. Functional respiratory deficiencies and decreased bacterial clearance ( Listeria monocytogenes) were found in mice treated with SWCNT. Equal doses of ultrafine carbon black particles or fine crystalline silica (SiO2) did not induce granulomas or alveolar wall thickening and caused a significantly weaker pulmonary inflammation and damage.
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Kumar Mangal, Chidambaram Shunmugam, Roy Anitha, and Thangavelu Lakshmi. "Inhibition of Nitric oxide Production and Nitric oxide Synthase Gene Expression in LPS Activated RAW 264 .7 Macrophages by Thyme oleoresin from Thymus vulgaris." Journal of Young Pharmacists 10, no. 4 (2018): 481–83. http://dx.doi.org/10.5530/jyp.2018.10.104.
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Jantová, Soňa, Dominika Topoľská, Michaela Janošková, Miroslav Pánik, and Viktor Milata. "Original Article. Study of the cytotoxic/toxic potential of the novel anticancer selenodiazoloquinolone on fibroblast cells and 3D skin model." Interdisciplinary Toxicology 9, no. 3-4 (2016): 106–12. http://dx.doi.org/10.1515/intox-2016-0014.
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Abstract The new synthetically prepared quinolone derivative 7-ethyl 9-ethyl-6-oxo-6,9-dihydro[1,2,5]selenadiazolo [3,4-h]quinoline-7-carboxylate (E2h) showed in our previous study cytotoxic effects towards tumor cells and immunomodulatory activities on RAW 264.7 cell line murine macrophages. E2h may have a potential use as a novel chemotherapeutic agent with immunomodulatory properties and the ability to induce apoptotic death of cancer cells. The aim of the present study was to examine the antiproliferative/cytotoxic activities of E2h on human non-cancer fibroblast BHNF-1 cells and reconstructed human epidermis EpiDerm™. Further the effects of E2h on tissue structure and morphology were examined. Cytotoxic/toxic studies showed that selenadiazoloquinolone is not toxic on normal human fibroblast cells BHNF-1 and dimensional skin constructs EpiDerm™. Evaluation of morphological changes in EpiDerm™ showed no change in the construction and morphology of skin tissue treated by E2h compared to control.
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Marzouk, Mohamed SA, Amira M. Gamal-Eldeen, Mona A. Mohamed, and Mortada M. El-Sayed. "Antioxidant and Anti-Proliferative Active Constituents of Tecoma Stans against Tumor Cell Lines." Natural Product Communications 1, no. 9 (2006): 1934578X0600100. http://dx.doi.org/10.1177/1934578x0600100908.
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A new phenylethanoid, 2-(3,4-dihydroxyphenyl)ethyl-,2-O-(6-deoxy-α-L-mannopyranosyl-,4-(3,4-dihydroxyphenyl)-2-propenoate)-β-D-glucopyranoside (3) and a novel monoterpene alkaloid, 5-hydroxy-skytanthine hydrochloride (8), along with eleven known compounds have been isolated from Tecoma stans Juss. fruits and flowers. 4-O-E-Caffeoyl-α-L-rhamnopyranosyl-(1′→3)-α/β-D-glucopyranose (1), E/Z-acetoside (2), isoacetoside (4), rutin (5), luteolin 7-O-β-D-neohespridoside (6), luteolin 7-O-β-D-glucopyranoside (7) and sucrose (9) were isolated from the fruits, while luteolin 7-O-β-D-glucuronopyranoside (10), diosmetin 7-O-β-D-glucuronopyranoside (11), diosmetin 7-O-β-D-glucopyranoside (12), diosmetin 7-O-β-D-glucuronopyranoside methyl ester (13), and 2 from the flowers. Their structures were determined on the basis of chemical and spectroscopic evidence. It was found that the extract of T. stans fruits and compounds 1, 2 and 4 possess strong scavenging activity to DPPH, peroxyl and hydroxyl radicals. Unlike 4, which potentially induced NO generation in bacterial lipopolysaccharide-stimulated Raw murine macrophages (RAW 264.7), the extract, and compounds 1, 2, and 8 significantly inhibited NO generation. The extract, and compounds 2 and 4 exhibited a cytotoxic effect on human hepatocarcinoma cells (Hep-G2), while the extract, 2 and 8 were potent growth inhibitors of human breast carcinoma (MCF-7). Also, 1 and 2 were remarkable growth inducers of human lymphoblastic leukemia cells (1301), whereas the extract, 2, and 8 stimulated the macrophage proliferation rate.
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Su, Yung-Shun, Ming-Der Wu, Jih-Jung Chen, et al. "Secondary Metabolites with Anti-Inflammatory Activities from One Actinobacteria Amycolatopsis taiwanensis." Molecules 26, no. 19 (2021): 5765. http://dx.doi.org/10.3390/molecules26195765.
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Phytochemical investigation and chromatographic separation of extracts from one new actinobacteria strain Amycolatopsis taiwanensis that was isolated from soil of Yilan township, in the north of Taiwan, led to the isolation of nine new compounds, amycolataiwanensins A–I (1–9, resp.), and one new natural product, namely amycolataiwanensin J (10). The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic-data analysis (1D- and 2D-NMR, MS, and UV) and comparison with literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Of the isolates, 3, 5, 7 and 8 exhibited potent anti-NO production activity, with IC50 values of 17.52, 12.31, 17.81 and 13.32 μM, respectively, compared to that of quercetin, an iNOS inhibitor with an IC50 value of 35.94 μM. This is the first report on indole metabolite from the genus Amycolatopsis.
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Su, Yung-Shun, Jih-Jung Chen, Ming-Jen Cheng, et al. "Saccharpiscinols A–C: Flavans with Potential Anti-Inflammatory Activities from One Actinobacteria Saccharomonospora piscinae." Molecules 26, no. 16 (2021): 4909. http://dx.doi.org/10.3390/molecules26164909.
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Phytochemical investigation and chromatographic separation of extracts from the actinobacteria strain Saccharomonospora piscinae that was isolated from dried fishpond sediment of Kouhu township, in the south of Taiwan, led to the isolation of three new compounds, saccharpiscinols A–C (1–3, respectively), and three new natural products, namely (2S)-5,7,3′,4′-tetrahydroxy-6,8-dimethylflavanone (4), methyl-4-hydroxy-2-methoxy-6-methylbenzoate (5), and (±)-7-acetyl-4,8-dihydroxy-6-methyl-1-tetralone (6). Compounds 4–6 were reported before as synthesized products, herein, they are reported from nature for the first time. The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic data analysis (1D- and 2D-NMR, MS, and UV) and comparison with literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Saccharpiscinol A showed inhibitory activities against LPS-induced NO production.
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Kishimoto, Chiharu, Hiroshi Kawamata, Shinya Sakai, Hiromichi Shinohara, and Hiroshi Ochiai. "Enhanced Production of Macrophage Inflammatory Protein 2 (MIP-2) by In Vitro and In Vivo Infections with Encephalomyocarditis Virus and Modulation of Myocarditis with an Antibody against MIP-2." Journal of Virology 75, no. 3 (2001): 1294–300. http://dx.doi.org/10.1128/jvi.75.3.1294-1300.2001.
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ABSTRACT Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein 2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine whether MIP-2 aggravates murine myocarditis. We examined (i) the MIP-2-producing activity of encephalomyocarditis (EMC) virus-infected cultured macrophages, (ii) serial plasma MIP-2 levels in EMC virus-induced mice by enzyme-linked immunosorbent assay, and (iii) the effects of antimouse MIP-2 monoclonal antibody (MAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264.7 macrophages. Five-week-old C3H/He mice were inoculated with EMC virus. Plasma MIP-2 levels were significantly elevated in mice on days 7 and 14 postinfection. Mice were injected subcutaneously with anti-MIP-2 MAb at 10 μg/day (group 2) or 100 μg/day (group 3) on days 0 to 5 and were observed until day 21. Uninfected control mice (group 1) were prepared. The survival rate was higher in the anti-MIP-2-treated group (group 3), but not in group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T-cell accumulation were less prominent in the anti-MIP-2 MAb-treated group, but not in group 2, compared to the level in the controls. MIP-2 is an important naturally occurring inflammatory cytokine in myocarditis, and anti-MIP-2 MAb treatment may prevent the inflammatory response.
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De Stefani, Chiara, Marzia Vasarri, Maria Cristina Salvatici, et al. "Microemulsions Enhance the In Vitro Antioxidant Activity of Oleanolic Acid in RAW 264.7 Cells." Pharmaceutics 14, no. 10 (2022): 2232. http://dx.doi.org/10.3390/pharmaceutics14102232.
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Oleanolic acid (OA) is the main triterpenic acid of olive leaves known for numerous pharmacological properties, including antioxidant activity. However, it is poorly soluble in water and consequently with low bioavailability, which limits its pharmacological application. Microemulsions (MEs) are dispersed systems consisting of two immiscible phases that promote rapid solubilization and absorption in the gastrointestinal tract. To improve both solubility and intestinal permeability of this molecule, OA has been formulated in two different microemulsions (ME-1 and ME-2). A solubility screening was carried out to select the ME components, and pseudoternary phase diagrams were constructed to evaluate the region of existence and select the appropriate amount of the constituents. ME-1 was prepared using Capmul PG-8/NF as the oily phase, and Transcutol and Tween 20 (7:3) as surfactants, while ME-2 contained Nigella oil and Isopropil myristate as the oily phase, and Transcutol HP and Cremophor EL (2:1) as surfactants. The OA solubility was increased by 1000-fold and 3000-fold in ME-1-OA and ME-2-OA, respectively. The MEs’ droplet size and the PdI were evaluated, and the stability was assessed for 8 weeks by monitoring chemical and physical parameters. The parallel artificial membrane permeability assay (PAMPA) also demonstrated an enhanced intestinal permeability of both OA formulations compared with free OA. The potential ability of both MEs to enhance the bioactivity of OA against LPS-induced oxidative stress in RAW 264.7 murine macrophages was also investigated. Overall, this study suggests that both MEs promote a bio-enhancement of the protective action of OA against the LPS-induced pro-oxidant stress in macrophages. Overall, this study suggests that MEs could be an interesting formulation to improve OA oral bioavailability with potential clinical applications.
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Limachi, Ivan, Mariela Gonzalez-Ramirez, Sophie Manner, et al. "Trichilianones A-D, Novel Cyclopropane-Type Limonoids from Trichilia adolfi." Molecules 26, no. 4 (2021): 1019. http://dx.doi.org/10.3390/molecules26041019.
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The fractionation of an ethanol extract of the bark of Trichilia adolfi yielded four novel limonoids (trichilinones A-D, 1–4), with five fused rings and related to the hortiolide-type limonoids. Starting with an ε-lactone, which is α,β-unsaturated in trichilinones A and D (1 and 4), attached to a tetrahydrofuran ring that is connected to an unusual bicyclo [5.1.0] hexane system, joined with a cyclopentanone with a 3-furanyl substituent [(2-oxo)-furan-(5H)-3-yl in trichilinone D (4)], the four compounds isolated display a new 7/5/3/5/5 limonoid ring system. Their structures were established based on extensive analysis of NMR spectroscopic data. As the crude extract possessed anti-leishmanial properties, the compounds were assayed for cytotoxic and anti-parasitic activities in vitro in murine macrophages cells (Raw 264.7) and leishmania promastigotes (L. amazoniensis and L. braziliensis), respectively. The compounds showed moderate cytotoxicity (approximately 70 μg/mL), but are not responsible for the leishmanicidal effect of the extract.
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Kratz, S. S., and R. J. Kurlander. "Characterization of the pattern of inflammatory cell influx and cytokine production during the murine host response to Listeria monocytogenes." Journal of Immunology 141, no. 2 (1988): 598–606. http://dx.doi.org/10.4049/jimmunol.141.2.598.
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Abstract To examine the physiologic mechanisms responsible for enhanced antibacterial activity during infection with Listeria monocytogenes (LM), we developed an in vitro assay for quantifying leukocyte anti-listerial activity (LAA) in spleen and bone marrow. When LAA was serially measured in C57B1/6 (B6) mice infected i.v. with LM, two distinct phases of response were observed. Splenic LAA increased four- to fivefold during the first 2 days after i.v. infusion of LM (from 2.4 +/- 1.8 U/spleen before infection to 11.8 +/- 2.4, p less than 0.01), dropped significantly on days 3 to 4, and increased again to similar levels from days 5 to 7. A fall in bone marrow activity from the 3.5 +/- 1.5 to 1.6 +/- 0.7 U/mouse (two femurs) coincided with the initial rise in splenocyte activity, and was followed by a gradual return to base line. Bacterial containment in vivo correlated well with splenic LAA in vitro. Carbonyl iron pretreatment of cells from both normal and LM-infected animals ablated LAA, suggesting the effectors were phagocytic. LAA in normal spleens was unaffected by 400 rad; LAA of normal marrow as well as splenocyte and marrow cell suspensions obtained 2 days after LM infection was markedly reduced by this dose of irradiation. Quantitative studies of spleen composition revealed a 10-fold increase in polymorphonuclear neutrophils between day 0 and day 2 followed by a marked decrease on day 3; this pattern closely resembled the changes in LAA observed during the same period. In contrast, splenic macrophage number did not increase from base line until after day 3. To look for evidence of changes in the efficiency of bacterial killing by phagocytes during infection, we calculated LAA/splenic phagocyte. The efficiency of killing increased threefold over base line within 1 day after LM infusion but we detected no additional increases later in infection. Because cytokines may have mediated some or all of the changes observed, we measured the capacity of splenocytes obtained at various times after infection to produce IL-2, TNF, and IFN-gamma in vitro. TNF activity increased in parallel with the first and second LAA peaks, whereas increases in IL-2 and IFN-gamma activity were associated only with the second.(ABSTRACT TRUNCATED AT 400 WORDS)
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Wypij, Magdalena, Tomasz Jędrzejewski, Maciej Ostrowski, Joanna Trzcińska, Mahendra Rai, and Patrycja Golińska. "Biogenic Silver Nanoparticles: Assessment of Their Cytotoxicity, Genotoxicity and Study of Capping Proteins." Molecules 25, no. 13 (2020): 3022. http://dx.doi.org/10.3390/molecules25133022.
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The development of nanotechnology in the last two decades has led to the use of silver nanoparticles (AgNPs) in various biomedical applications, including antimicrobial, anti-inflammatory, and anticancer therapies. However, the potential of the medical application of AgNPs depends on the safety of their use. In this work, we assessed the in vitro cytotoxicity and genotoxicity of silver nanoparticles and identified biomolecules covering AgNPs synthesized from actinobacterial strain SH11. The cytotoxicity of AgNPs against MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 was studied by MTT assay, cell LDH (lactate dehydrogenase) release, and the measurement of ROS (reactive oxygen species) level while genotoxicity in Salmonella typhimurium cells was testing using the Ames test. The in vitro analysis showed that the tested nanoparticles demonstrated dose-dependent cytotoxicity against RAW 264.6 macrophages and MCF-7 breast cancer cells. Moreover, biosynthesized AgNPs did not show a mutagenic effect of S. typhimurium. The analyses and identification of biomolecules present on the surface of silver nanoparticles showed that they were associated with proteins. The SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) analysis revealed the presence of 34 and 43 kDa protein bands. The identification of proteins performed by using LC-MS/MS (liquid chromatography with tandem mass spectrometry) demonstrated their highest homology to bacterial porins. Capping biomolecules of natural origin may be involved in the synthesis process of AgNPs or may be responsible for their stabilization. Moreover, the presence of natural proteins on the surface of bionanoparticles eliminates the postproduction steps of capping which is necessary for chemical synthesis to obtain the stable nanostructures required for application in medicine.
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Ma, Jonathan, Apparao B. Kummarapurugu, Adam Hawkridge, Shobha Ghosh, Shuo Zheng, and Judith A. Voynow. "Neutrophil elastase-regulated macrophage sheddome/secretome and phagocytic failure." American Journal of Physiology-Lung Cellular and Molecular Physiology 321, no. 3 (2021): L555—L565. http://dx.doi.org/10.1152/ajplung.00499.2019.
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Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.
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Bonamin, Leoni Villano, Fabiana Rodrigues Santana, Luciane Costa Dalboni, et al. "Effects of Antimomium crudum 30cH and 200cH on the macrophage – Leishmania (L) amazonensis relation in vitro." International Journal of High Dilution Research - ISSN 1982-6206 15, no. 4 (2021): 7–8. http://dx.doi.org/10.51910/ijhdr.v15i4.836.
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Background: Leishmaniasis is a chronic skin and systemic disease, whose treatment is related to important side effects and loss of life quality. In previous results [1], mice treated with Antimonium crudum (AC) 30cH presented reduction in local inflammatory process and modulation of B1 cell-phagocyte differentiation and migration, but also increase of free amastigotes number among the surrounding tissue. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with AC 30cH and 200cH, in different times. Methodology: The morpho-functional features of macrophages (spreading, phagocytosis and oxidative activity), the number of free promastigotes in the supernatant and the cytokines measurement were evaluated. The spreading and phagocytosis assays were performed in quadruplicate, in three experiments, resulting in 12 datapoints for each dilution/time. Kruskal Wallis (for no parametric variables) and two-way ANOVA test (for parametric variables) were used to verify time to time differences and the interaction between treatment and time respectively, being p≤0.05 considered significant. Animals were not used in this study. Results: The treatment with AC 30cH and AC 200cH resulted in significant but transitory increase in spreading and phagocytosis activity after 2 to 24 hours of incubation, followed by increase in the number of free promastigotes in the supernatant (in AC 30cH treatment) and decrease in CD86 expression (in AC 200cH treatment). GMCSF, alpha-INF, IL1, IL6, IL10 and IL12 were reduced after 48 to 72 hours and CCL4 and RANTES were reduced after 120H, for both potencies. Two peaks of CCL2 were seen in Leishmania sp infected macrophages, at 24 and 120 hours, but only AC30cH inhibited them. A peak of VEGF was observed after 120 hours following the treatment with AC 200cH, together with the increase in the number of multinucleated cells. The morphology of macrophages at fluorescence microscopy after the staining with acridine orange in fresh unfixed cells showed severe reduction of acid vacuoles in AC 30CH treated cells at 2 hours of parasite-macrophage interaction, revealing possible macrophage anergy. No difference in peroxide / NO production and in apoptosis percentage of free promastigotes was seen among groups, in all evaluated times. Conclusions: Both potencies were able to decrease most of cytokines production, specially CCL2 in AC 30cH treated cells, which explains the inhibition of monocyte migration seen in vivo [1]. The late peak of VEGF observed in AC 200cH treated cells suggests a M1 - M2 polarization, whose biological meaning is still under discussion.
 
 Acknowledgements: CAPES, UNIP, FAPESP (2014/00967-1)
  
 References
 Rodrigues de Santana F, de Paula Coelho C, Cardoso TN, Perez Hurtado EC, Roberti Benites N, Dalastra Laurenti M, Villano Bonamin L. Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH. Homeopathy, 2014;103(4):264-74. doi: 10.1016/j.homp.2014.08.006.
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Tham, Sin Mun, Juwita N. Rahmat, Edmund Chiong, Qinghui Wu, Kesavan Esuvaranathan та Ratha Mahendran. "Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model". Biomedicines 9, № 12 (2021): 1766. http://dx.doi.org/10.3390/biomedicines9121766.
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This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12–16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.
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Casas-Arrojo, Virginia, María de los Ángeles Arrojo Agudo, Casimiro Cárdenas García, et al. "Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs." Pharmaceuticals 15, no. 11 (2022): 1379. http://dx.doi.org/10.3390/ph15111379.
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The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL−1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL−1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography–tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.
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Brindisi, Matteo, Chouaha Bouzidi, Luca Frattaruolo, et al. "Chemical Profile, Antioxidant, Anti-Inflammatory, and Anti-Cancer Effects of Italian Salvia rosmarinus Spenn. Methanol Leaves Extracts." Antioxidants 9, no. 9 (2020): 826. http://dx.doi.org/10.3390/antiox9090826.
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In this study, we evaluated and compared the chemical composition, the antioxidant, anti-inflammatory, and anti-proliferative effects of four methanol extracts (R1–R4), of Salvia rosmarinus Spenn. in two different sites of Southern Italy obtained by maceration or ultrasound-assisted extraction. Extracts of S. rosmarinus collected on the Ionian coast are indicated with the abbreviations R1 (maceration) and R2 (ultrasound-assisted extraction). Extracts of S. rosmarinus collected on the Tyrrhenian coast are indicated with the abbreviations R3 (maceration) and R4 (ultrasound-assisted extraction). The chemical composition was analyzed using High Pressure liquid chromatography–Diod-Array detection–Electrospray ionization–Quadrupole–Mass Spectroscopy (HPLC-DAD-ESI-Q-MS). The antioxidant activity was analyzed by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene bleaching, and Ferric Reducing Antioxidant Power (FRAP) assays. Antioxidant features were also assessed in lipopolysaccharide (LPS)-stimulated RAW-264.7 murine macrophages, evaluating Reactive Oxygen Species (ROS) production; in the same experimental model, the anti-inflammatory activity of the extracts was investigated. Interestingly, all extracts displayed antioxidant and anti-inflammatory properties. They exhibited significative nitrite production inhibitory activity, whith IC50 values ranging from 3.46 to 5.53 µg/mL, without impairing cell viability. The anti-inflammatory activity was also investigated by Western Blotting and immunofluorescence assay, highlighting the R3 and R4 extracts ability to reduce NF-κB translocation, as well as to disrupt the MAPKs signaling pathway. Extracts exhibited both potential anti-proliferative activity on breast cancer cells, inducing apoptosis, without affecting non-tumorigenic cells, and the ability to inhibit MDA-MB-231 cells’ motility. Finally, the rosemary extracts treatment significantly reduced the power of conditioned media, from MCF-7 or MDA-MB-231 cells to induce nitrite production on RAW 264.7 cells, confirming their promising anti-inflammatory activity.
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de Oliveira, Jonatas Rafael, Daiane de Jesus, Leandro Wagner Figueira, et al. "Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells." Experimental Biology and Medicine 242, no. 6 (2017): 625–34. http://dx.doi.org/10.1177/1535370216688571.
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R. officinalis L. is an aromatic plant commonly used as condiment and for medicinal purposes. Biological activities of its extract were evaluated in this study, as antimicrobial effect on mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory capacity, and genotoxicity. Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed of C. albicans with each bacterium were formed in microplates during 48 h and exposed for 5 min to R. officinalis L. extract (200 mg/mL). Its cytotoxic effect was examined on murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7), and cervical carcinoma cells (HeLa) after exposure to different concentrations of the extract, analyzed by MTT, neutral red (NR), and crystal violet (CV) assays. The anti-inflammatory activity was evaluated on RAW 264.7 non-stimulated or stimulated by lipopolysaccharide (LPS) from Escherichia coli and treated with different concentrations of the extract for 24 h. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) were quantified by ELISA. Genotoxicity was verified by the frequency of micronuclei (MN) at 1000 cells after exposure to concentrations of the extract for 24 h. Data were analyzed by T-Test or ANOVA and Tukey Test ( P ≤ 0.05). Thus, significant reductions in colony forming units per milliliter (CFU/mL) were observed in all biofilms. Regarding the cells, it was observed that concentrations ≤ 50 mg/mL provided cell viability of above 50%. Production of proinflammatory cytokines in the treated groups was similar or lower compared to the control group. The MN frequency in the groups exposed to extract was similar or less than the untreated group. It was shown that R. officinalis L. extract was effective on mono- and polymicrobial biofilms; it also provided cell viability of above 50% (at ≤ 50 mg/mL), showed anti-inflammatory effect, and was not genotoxic. Impact statement Rosmarinus officinalis L. extract effectively contributed to in vitro control of important species of microorganisms such as Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa in mono- and polymicrobial biofilms that are responsible for several infections in oral cavity as in other regions of the body. Furthermore, this extract promoted also cell viability above 50% at concentrations ≤ 50 mg/mL, excellent anti-inflammatory effect, showing inhibition or reduction of the synthesis of proinflammatory cytokines, being also non-genotoxic to cell lines studied. Thus, this extract may be a promising therapeutic agent that can be added in some medical and dental formulations such as toothpastes, mouthwashes, irrigating root canals, ointments, soaps, in order to control pathogenic microorganisms and biofilms, with anti-inflammatory effect and absence of cytotoxic and genotoxic.
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Collery, Philippe, Didier Desmaële, Adhikesavan Harikrishnan, and Vijay Veena. "Remarkable Effects of a Rhenium(I)-diselenoether Drug on the Production of Cathepsins B and S by Macrophages and Their Polarizations." Current Pharmaceutical Design 29 (October 18, 2023). http://dx.doi.org/10.2174/0113816128268963231013074433.
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Background/Objective: Tumor-associated macrophages (TAMs) produce an excessive amount of cysteine proteases, and we aimed to study the effects of anticancer rhenium(I)-diselenoether (Re-diSe) on the production of cathepsins B and S by macrophages. We investigated the effect of Re-diSe on lipopolysaccharides (LPS) induced M1 macrophages, or by interleukin 6 (IL-6) induced M2 macrophages. Methods: Non-stimulated or prestimulated murine Raw 264 or human THP-1 macrophages were exposed to increasing concentrations of the drug (5, 10, 20, 50 and 100 μM) and viability was assayed by the MTT assay. The amount of cysteine proteases was evaluated by ELISA tests, the number of M1 and M2 macrophages by the expression of CD80 or CD206 biomarkers. The binding of Re-diSe with GSH as a model thiol-containing protein was studied by mass spectrometry. Results: A dose-dependent decrease in cathepsins B and S was observed in M1 macrophages. There was no effect in non-stimulated cells. The drug induced a dramatic dose-dependent increase in M1 expression in both cells, significantly decreased the M2 expression in Raw 264 and had no effect in non-stimulated macrophages. The binding of the Re atom with the thiols was clearly demonstrated. Conclusion: The increase in the number of M1 and a decrease in M2 macrophages treated by Re-diSe could be related to the decrease in cysteine proteases upon binding of their thiol residues with the Re atom.
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Madden, Abbie J., Madison D. Krehbiel, and Stephen L. Clarke. "Garlic‐derived Compounds Increase Expression of ABCA1 mRNA in RAW 264.7 Murine Macrophages." FASEB Journal 31, S1 (2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.973.1.
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ObjectiveConsiderable interest remains in the role of bioactive components in garlic. While extensive studies have explored the potential of these compounds to initiate epigenetic modification beneficial in cancer treatment, mechanism(s) underlying the effects of garlic on cholesterol metabolism remain unclear. Of particular interest is the ATP‐binding cassette transporter A1 (ABCA1), a protein that facilitates reverse cholesterol efflux from macrophages to high density lipoproteins (HDL). Efflux can prevent macrophages from becoming pro‐atherogenic foam cells, thereby reducing the risk of cardiovascular disease. The purpose of this study was to investigate the effects in mouse macrophages of garlic compounds allyl mercaptan (AM) and diallyl disulfide (DADS) on expression of ABCA1 compared with Trichostatin A (TSA), a drug known to induce ABCA1 via histone modification.MethodsRAW 264.7 cells were treated for 6 hours with 1.2 mM AM, 200 μM DADS, or 10 ng/mL TSA. Total RNA was isolated and reverse transcribed, and ABCA1 mRNA expression was evaluated by quantitative polymerase chain reaction (qPCR). Analysis was done using the 2−ΔΔCt method, with cyclophilin B as the invariant control.ResultsqPCR analysis revealed a 11‐ and 7‐fold increase of ABCA1 mRNA expression compared to control after treatment of AM and DADS, respectively. In comparison, TSA induced expression by 3‐fold. Studies are ongoing to test effects of garlic treatment on cholesterol synthesis and efflux, as well as potential molecular mechanism(s) through which these compounds regulate ABCA1 expression.ConclusionAM and DADS induce mRNA expression of ABCA1 in RAW 264.7 macrophages. These compounds may play an important protective role in promoting reverse cholesterol efflux, which can subsequently benefit overall cholesterol homeostasis.
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Wang, Ning-Ning, Chun-Yu Liu, Tian Wang, Yue-Lan Li, Ke Xu, and Hong-Xiang Lou. "Two New Quinazoline Derivatives from the Moss Endophytic Fungus Aspergillus sp. and Their Anti-inflammatory Activity." Natural Products and Bioprospecting, November 21, 2020. http://dx.doi.org/10.1007/s13659-020-00287-5.
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Abstract Two new quinazoline derivatives versicomides E (1) and F (2), and 10 known compounds (3–12) were isolated from the moss endophytic fungus Aspergillus sp. Their structures were determined on the basis of extensive spectroscopic data analysis and ECD calculations. Among them, the compound 7 (6-hydroxy-3-methoxyviridicatin) was first reported as a natural product. Inhibition on LPS-induced NO production in RAW 264.7 murine macrophages found that compounds 5, 7 and 8 showed significant inhibitory effects on NO production, with IC50 values of 49.85, 22.14 and 46.02 μM respectively. Graphic Abstract
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Ortmann, Weronika, Anna Such, and Elzbieta Kolaczkowska. "Impact of microparticles released during murine systemic inflammation on macrophage activity and reactive nitrogen species regulation." Immunologic Research, November 27, 2023. http://dx.doi.org/10.1007/s12026-023-09436-7.
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AbstractMicroparticles (MPs) packaged with numerous bioactive molecules are essential vehicles in cellular communication in various pathological conditions, including systemic inflammation, Whereas MPs are studied mostly upon isolation, their detection in vivo is limited. Impact of MPs might depend on target cell type and cargo they carry; thus herein, we aimed at verifying MPs’ impact on macrophages. Unlike neutrophils, monocytes/macrophages are rather inactive during sepsis, and we hypothesized this might be at least partially controlled by MPs. For the above reasons, we focused on the detection of MPs with intravital microscopy (IVM) and report the presence of putative neutrophil-derived MPs in the vasculature of cremaster muscle of endotoxemic mice. Subsequently, we characterized MPs isolated not only from their blood but also from the peritoneal cavity and observed differences in their size, concentration, and cargo. Such MPs were then used to study their impact on RAW 264.7 macrophage cell line performance (cell viability/activity, cytokines, oxygen, and nitrogen reactive species). Addition of MPs to macrophages with or without co-stimulation with lipopolysaccharide did not affect respiratory burst, somewhat decreased mitochondrial activity but increased inducible nitric oxide synthase (iNOS) expression, and NO production especially in case of plasma-derived MPs. The latter MPs carried more iNOS-controlling ceruloplasmin than those discharged into the peritoneal cavity. We conclude that MPs can be detected in vivo with IVM and their cellular origin identified. They are heterogeneous in nature depending on the site of their release. Consequently, microparticles released during systemic inflammation to various body compartments differentially affect macrophages.
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ah Kim, Hyun, Miri Hyun, Jiyeon Lee, Jin Kyung Kim, and wonki baek. "282. Resistance to caspase-1 dependent pyroptosis of hypervirulent K. pneumoniae." Open Forum Infectious Diseases 10, Supplement_2 (2023). http://dx.doi.org/10.1093/ofid/ofad500.354.
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Abstract Background Hypervirulent K. pneumoniae(Hv Kp) has emerged as a clinically significant global pathogen in the last decade. However, the host immune responses of the macrophages during hvKp infection are largely unknown. In the present study, we aimed to compare the cytotoxic effects of hvKp and classical K. pneumoniae (cKp) in murine macrophages. Methods Bacterial strains are obtained from clinical samples who infected by K. pneumoniae. The rmpA and iutA positive strains with mucoid phenotype were defined as hypervirulent K. pneumoniae. Raw 264.7 cell and BMDM(bone marrow derived macrophage) cell were used for experiments. Wild-type C57BL/6 female mice and Casp1-/- female mice (age, 7–9 weeks) were used. Cytotoxicity after 4 hours of infection, MTS and LDH release was measured. FACS scan after PI dyed was performed for cell cycle analysis and cytotoxicity each MOI(Multiplicity of infection) concentration. Western blot analysis of caspase-1 and caspase-1 inhibitor were used. IL-1β was tested with ELISA. Results We found that the activation of caspase-1 (Casp1)-dependent pyroptosis was higher in cKp-infected macrophages compared with that in hvKp-infected macrophages. In caspase-1 deficiency macrophages, pyroptosis diminished during infection. Both hvKp and cKp strains led to nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome formation and lysosomal cathepsin B activation, thus resulting in pyroptosis. Compared with the cKp strain, the hvKp strain inhibited these phenomena in murine macrophages.Figure 1.HvKp and cKp strains induce pyroptotic cell death in the RAW264.7 murine macrophage cell line.Figure 2.BMDMs were differentiated from WT and Casp1-/- mice and infected with the hvKp or cKp strains. Conclusion In this study, compared to classical strains, it was confirmed that cells infected with hv Kp survived without apoptosis. HvKp infection resulted in different levels of pyroptosis via the activation of cathepsin B-NLRP3-caspase-1 in murine macrophage. Therefore, the manipulation of pyroptotic cell death is a potential target for host response during hvKp infection in macrophages. Disclosures All Authors: No reported disclosures
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Caldari-Torres, Cristina, and Jordan Beck. "Effects of co-incubation of LPS-stimulated RAW 264.7 macrophages on leptin production by 3T3-L1 adipocytes: a method for co-incubating distinct adipose tissue cell lines." Bulletin of the National Research Centre 46, no. 1 (2022). http://dx.doi.org/10.1186/s42269-022-00747-7.
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Abstract Background Adipose tissue is a major endocrine organ capable of releasing inflammatory adipokines that are linked to changes occurring in the overfed state, where tissue remodeling results in hypertrophic adipocytes that recruit monocytes to infiltrate the tissue and take on an inflammatory phenotype. Increases in macrophage-specific inflammatory mediator levels contribute to the inflamed state and worsen the inflammatory loop between the macrophages and adipocytes. Although most inflammatory adipokines are released by macrophages, adipocytes can also release immunomodulatory adipokines, such as leptin. The objective of this research was to determine if co-incubation of activated macrophages with mature adipocytes, using transwell inserts, affected adipocyte leptin release. We also examined if there were differences in levels of cell-secreted products quantified in cell-conditioned media collected from macrophage-containing (transwell insert) and adipocyte-containing (well) compartments. Methods Mature adipocytes were co-incubated with control and lipopolysaccharide-stimulated (0.01 mg/ml) murine macrophages, and nitric oxide, interleukin-6, and leptin levels were quantified in the cell-conditioned media from both compartments. Results Activation status of the macrophages did not affect leptin release by the adipocytes. We observed higher amounts of leptin in wells compared to transwells. Nitric oxide and interleukin-6 levels were similar between transwells and wells, suggesting that these adipokines travel through the transwell inserts and are reaching equilibrium between the two compartments. Conclusion Our results suggest that co-incubating activated macrophages and adipocytes using transwell inserts can result in distinct microenvironments in the different cellular compartments and that separate sampling of these compartments is required to detect the subtle signaling dynamics that exist between these cells.
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Roth, Paige, Jone Stanley, Ana Chamoun-Emanuelli, Canaan Whitfield-Cargile, and Michelle Coleman. "Fecal extract from obese horses induces an inflammatory response by murine macrophages in vitro." American Journal of Veterinary Research, February 2, 2022, 1–7. http://dx.doi.org/10.2460/ajvr.21.02.0024.
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Abstract OBJECTIVE To compare the inflammatory response of murine macrophages exposed to the enteric microbiome of obese horses versus nonobese horses. SAMPLE Fecal samples from 12 obese horses (body condition score ≥ 7/9) and 12 nonobese horses (body condition score 4 to 5/9) with similar dietary management. PROCEDURES Fecal supernatant was prepared from frozen fecal samples. RAW 264.7 macrophage cells were exposed to the fecal extract. Inflammatory cytokine (interleukin-1β, tumor necrosis factor-α, and interleukin-6) gene expression was quantified via real-time quantitative reverse transcription PCR assay, and cytokine concentration was quantified via ELISA. Lipopolysaccharide was evaluated in fecal extract via chromo-limulus amoebocyte lysate assay. RESULTS Compared with fecal extracts from nonobese horses, fecal extracts from obese horses presented higher concentrations of lipopolysaccharide and induced a heightened expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 from macrophages. CLINICAL RELEVANCE The increased levels of inflammatory markers induced in murine macrophages by the microbiome of obese horses in vitro suggested important differences in the enteric microbial composition of these horses, compared with nonobese horses. Overall, this study showed that the microbiome may play a role in mediating an inflammatory response within the gastrointestinal tract of obese horses. Mechanisms of obesity in the horse have not been fully elucidated. Improved understanding of the pathophysiology of disease will guide future research into potential diagnostic and therapeutic interventions for equine obesity.