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Maca (Lepidium meyenii) is a tuber root plant and belong to Brassicaceae family and recently used as a supplement in human and animal nutrition. In this study, it was aimed to investigate the cytotoxicity of two solutions (aqueous and ethanol) of maca root powder by digital image analysis. Maca powder was mixed in ultra distilled water and ethanol (1:2 w/v) for 24 h at 4 °C. The mixtures were centrifuged and the supernatants were ten-fold diluted for cytotoxic analysis of Raw 264.7 murine macrophage cells. After seeded the cell cultures in microplates, ten-fold dilutions (from 10-1 to 10-7 ) of both maca solutions were added as six replicates for 24 h. While the aqueous maca solution increased the number of dead cells at 10-1 (50 mg mL-1 ), the ethanolic solution statistically increased the number of dead cells at 10-1 , 10-2 and 10-3 (50, 5 and 0.5 mg mL-1 ) (P

Plant-derived exosome-like nanoparticles (PDENs) are small vesicles released by multivesicular bodies mainly to communicate between cells and regulate immunity against pathogen attack. Current studies have reported that PDENs could modulate gene expression in a cross-kingdom fashion. Therefore, PDENs could be a potential future functional food ingredient as their cross-kingdom communication abilities were reported to exert multiple health benefits. Macrophage and other cells have been reported to absorb PDENs in a manner regulated by the membrane lipid and protein profile and the intactness of the PDENs lipid bilayer. PDENs could be extracted from plant materials by various techniques such as ultracentrifugation, immunoaffinity, size-based isolation, and precipitation, though each method has its pros and cons. PDENs mainly contain lipid, protein, and genetic materials, mainly micro RNAs, which could exert multiple health benefits and functionalities when consumed in sufficient amounts. However, most studies on the health functionalities of PDENs were conducted through in-vitro and in-vivo studies, and its potency to be used as a functional ingredient remains a question as PDENs are sensitive to storage and processing condition and requires costly extraction method. This concise review features various exosome extraction methods, contents of PDENs and their roles, the health functionalities of PDENs, and its potency as a functional food ingredient.

Blaps rynchopetera Fairmaire has a long history of use as a folk medicine in China for treating fever, cough, gastritis, boils, and tumors. In the present study, the content analyses, monosaccharide composition analyses, and the macrophage phagocytic activity of rynchopetera polysaccharides (RPS) were reported. B. rhynchoptera is rich in polysaccharides (content value 3.97%). Through PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization and high performance liquid chromatography (HPLC) testing, the results showed that RPS consist of 8 known monosaccharides, including D-mannose (Man), Rhamnose (Rha), D-glucuronic acid (GlcUA), D-galacturonic acid (GalUA), D-glucose (Glc), D-galactose (Gal), Arabinose (Ara), and Fucose (Fuc), with the total content of 171.70 mg g−1 and Glc has the highest content of 45.40 mg g−1. The phagocytic ability of mouse peritoneal macrophages was investigated after RPS stimulating alone and combined with lipopolysaccharide (LPS). RPS played an important role in the engulfment of mouse peritoneal macrophages and can significantly enhance the phagocytic ability of macrophages. However, no synergistic effects were observed when RPS combined with LPS.

X-ray microscopy is increasingly used in biology, but in most cases only in a qualitative way. We present here a 3D correlative cryo X-ray microscopy approach suited for the quantification of molar concentrations and structure in native samples at nanometer scale. The multimodal approach combines X-ray fluorescence and X-ray holographic nanotomography on "thick" frozen-hydrated cells. The quantitativeness of the X-ray fluorescence reconstruction is improved by estimating the self-attenuation from the 3D holography reconstruction. Applied to complex macrophage cells, we extract the quantification of major and minor elements heavier than phosphorus, as well as the density, in the different organelles. The intracellular landscape shows remarkable elemental differences. This novel analytical microscopy approach will be of particular interest to investigate complex biological and chemical systems in their native environment.

Background: According to the “silica hypothesis” to explain the action of homeopathic medicines, complex nanoparticles involving the silica extracted from the walls of the glass vial during succussions would be needed to act on target cells. Aims: We aimed to know if suspended particles of silica found in Arsenicum album (AA) could be associated to its biological effects in vitro. Methodology: RAW 264.7 macrophages were co-cultured with yeast (Saccharomyces cerevisiae) to promote phagocytosis. During this process, the cells were exposed to AA in different homeopathic dilutions (30K, 6cH and 200cH) prepared in both, glass or silica-fee plastic vials. All dilutions were prepared from the same 3cH matrix. Since parallel experiments showed random stimulating and inhibiting effects of homeopathic lactose, untreated cells and water were used as controls. The macrophage activity was measured by digital histometry (Metamorph®), reflecting the cell spreading and phagocytosis. A complementary analysis using the acridine orange stain in real time fluorescence microscopy was also performed to evaluate the organization of phago-lysosomes inside the cells. All experiments were performed in triplicate. Microparticles present in the medicines were identified in an EDS (energy dispersive x-ray detector) system (JEOL JMS 6510), according to their chemical elements composition. The morphology of particles was not considered for analysis, since it was considered irrelevant. Results: Experiment one: AA 30K and pure water, when prepared in plastic vials, were able to increase macrophage activity (p≤0.05), but the same result was not seen in cells treated with AA 30K or pure water stocked in glass. Experiment two: AA 200cH prepared in plastic vials also lead to significant increase of macrophage spreading (p≤0.05) in relation to the controls, but no effect was seen in the samples prepared in glass vials. Experiment three: No difference was observed in AA 6cH treated cells in relation to the controls. The morphology of the cells under fluorescence microscopy exhibited increase in lysosome activity in AA 200cH prepared in both plastic and glass vials, in relation to the untreated control. The EDS analysis has shown that only medicines prepared in glass vials contained suspended silica particles. Conclusions: Taken the data together, the unspecific effects on macrophage spreading after treatment with homeopathic AA 30K, 200cH and water prepared in plastic vials, added to the absence of silica particles in these preparations and the increase of lysosomal activity only seen in AA 200cH, independently of the vial nature, corroborate that silica might not be a mandatory condition to justify the biological effects of homeopathic medicines. Additionally, the existence of putative contaminants released from plastic vials during the medicine manipulation deserves further studies. Acknowledgements: CAPES, UNIP, ABFH

A 70-days feeding trial was performed to determine the effect of diets with whole plant-origin proteins added with different ratios of taurine:methionine on the growth, macrophage burst activity and antioxidant capacity of rainbow trout (Oncorhynchus mykiss) fingerlings. Triplicated groups of 70 fingerlings of an initial weight of 0.54±0.1 g (mean±±SD) were fed diets with soy protein isolate and Spirulina powder as protein sources (46% crude protein) and added (10 g/kg diet) with different taurine and methionine ratios: 0.0 and 10.0 (diet T0/M100), 2.5 and 7.5 (diet T25/M75), 5.0 and 5.0 (diet T50/M50), 7.5 and 2.5 (diet T75/M25) and 10.0 and 0.0 (diet T100/M0), respectively. At the end of the trial, growth performance, lipid and protein contents in liver and muscle, macrophage burst activity and liver antioxidant activity, were determined. The growth performance, macrophage burst activity and antioxidant activity were improved as the taurine increased in the diets. The ratio of 7.5 and 2.5 g/kg of taurine:methionine in diets with SPI and Spirulina powder as protein sources (diet T75/M25), seems to be the best inclusion for rainbow trout fingerlings., Highlights • Several ratios of taurine:methionine for rainbow trour fed while plant-protein diets. • Taurine improved antioxidant capacity of heptocites and burst activity of macropahges. • 10 g/kg of taurine:methionine inclusion is optimal for rainbow trout fed whole plant protein diets.

In vitro studies are a first step toward understanding the biological effects of combustion-derived particulate matter (cdPM). A vast majority of studies expose cells to cdPM suspensions, which requires a method to collect cdPM and suspend it in an aqueous media. The consequences of different particle collection methods on particle physiochemical properties and resulting biological responses are not fully understood. This study investigated the effect of two common approaches (collection on a filter and a cold plate) and one relatively new (direct bubbling in DI water) approach to particle collection. The three approaches yielded cdPM with differences in particle size distribution, surface area, composition, and oxidative potential. The directly bubbled sample retained the smallest sized particles and the bimodal distribution observed in the gas-phase. The bubbled sample contained ∼50% of its mass as dissolved species and lower molecular weight compounds, not found in the other two samples. These differences in the cdPM properties affected the biological responses in THP-1 cells. The bubbled sample showed greater oxidative potential and cellular reactive oxygen species. The scraped sample induced the greatest TNFα secretion. These findings have implications for in vitro studies of air pollution and for efforts to better understand the underlying mechanisms.

Fish collagen have the potential to be used in biomedical fields due to the biocompatible and biodegradable features. However, how hydrolyzed fish collagen regulate human macrophage has not been clearly unraveled. The aim of the current study is to investigate the effects of hydrolyzed fish collagen on the polarization state of human macrophages. The cell viability of human macrophages treated by hydrolyzed fish collagen were determined using CCK-8 assay. The polarization of human macrophages treated by hydrolyzed fish collagen were detected by quantitative real-time polymerase chain reaction (qRT-PCR). it was found that 0.25,0.5 and 1mg/ml hydrolyzed fish collagen did not alter the cell viability of human macrophages. Further, the data showed that hydrolyzed fish collagen inhibited the expression of M1 macrophage marker IL-ip and TNF-a while enhanced the expression of M2 macrophage marker arginase 1(Arg1) and IL-10. Taken together, the results indicated that treatment with hydrolyzed fish collagen result in a significant shift towards an M2 phenotype for human macrophages.

Dendrobium spp. are precious medicinal plants, used in China for thousands of years as health foods and nutrients. Polysaccharides are the main effective ingredients in Dendrobium plants. In this study, the chemical characteristics and the effects of crude polysaccharides (CPs) from five species of Dendrobium on macrophage function were investigated and compared in vitro for the first time. Chemical characteristic studies showed that CPs from different species of Dendrobium were diverse, displaying widely varied Mw distributions and molar ratios of monosaccharides. Their effects on macrophage functions, such as promoting phagocytosis, release of NO and cytokines IL-1α, IL-6, IL-10 and TNF-α, were also different. Moreover, CPs from D. officinale, especially collected from Yunnan Province, exerted the strongest immunomodulatory activities and could be explored as a novel potential functional food. The diverse chemical characteristics of CPs from different species of Dendrobium might contribute to their varied effects on macrophage functions, which should be further investigated.

Two experiments were conducted to study the interaction between dietary lutein and fat levels in broiler chicks hatched from lutein depleted (Experiment I) and repleted (Experiment II) eggs. In both experiments, a 2 x 3 factorial arrangement of treatments resulted in six dietary treatments (fat at 3% and 6% and lutein at 0, 25 and 50 mg/kg feed) that were fed for 23 days to 18 birds per treatment (in three replications). In Experiment I, the anti-dinitrophenyl-keyhole-lympet-hemocyanin (anti-DNP-KLM) serum antibody response at day 22 and macrophage phagocytotic index at day 17 did not differ among treatment groups (p > 0.05). The concavalin A and phytohaemagglutinin-P lymphocyte proliferation index at day 19 was greater in birds fed 50 mg of lutein and 3% fat than in birds fed all other diets (p < 0.05). Independent of the level of dietary fat, dietary lutein increased macrophage (day 23) nitrite production measured 46 h after in vitro stimulation with LPS (p < 0.05). Among the birds fed lutein at 25 or 50 mg/kg feed, birds fed 3% fat had higher LPS-induced nitrite production compared to the birds fed 6% fat after 46 (p = 0.014) or 70 h (p < 0.001). In Experiment II, macrophage nitrite production was measured at 54 h after LPS stimulation on days 11, 15, 19 and 23. An interaction between dietary lutein and fat levels on nitrite production was observed on day 19 (p = 0.012), where macrophages from birds fed 0 mg lutein and 3% fat had the highest nitrite production (p = 0.012). Macrophages from birds fed lutein at 25 and 50 mg/kg diet and 3% fat had higher (p = 0.012) nitrite production than birds fed 6% fat. Thus, in birds hatched from lutein deplete and replete eggs, modulation of macrophage nitrite production by lutein is dependent on the level of dietary fat.

Previous reports by others establish that particle surface area is related to a change in macrophage function as measured by the ability to clear particles from the alveolar spaces. However, for nanoparticles the relation may not be strictly due to surface chemistry: The cumulative projected area of the particles may reflect the degree to which the inner or outer surface of the macrophage is shielded from other objects or molecules. We apply this alternative interpretation to in vitro measurements of macrophage uptake of 26-nm-diameter fluorescent beads and to in vivo data presented in a classic inhalation toxicology paper on nano-sized TiO2 particles. In their paper, Oberdörster et al. (Environ. Health Perspect. 102[suppl. 5]:173-179, 1994) reported that following inhalation exposure to 20-nm or 250-nm TiO2 particles, the half-times for alveolar clearance of polystyrene test particles were proportional to square centimeters of TiO2 particle surface per million macrophages; macrophage toxicity from TiO2 particle surface was assumed to be the cause of the decrease in the clearance rate of polystyrene test particles. When TiO2 particle projected area was incorporated into the in vivo macrophage dosimetry calculations, particle projected areas ranged in value from covering only a fraction (0.1) of the macrophage surface to covering the cell surface 4 times over. The observed decrease in macrophage mediated alveolar clearance of polystyrene test particles was directly related to the potential for TiO2 particles to mask the surface of the macrophage-a possibility that was visualized in vitro with confocal laser scanning microscopy.

Several studies have tackled the evaluation of the biocompatibility of nanomaterials in cell and tissue. Nonetheless to date, a quantitative technique for the assessment of the total intracellular nanocarrier dose administered has not been introduced. In this paper we develop two rapid and sensitive assays for the measurement of internalized nanomaterials in macrophages to be applied as vehicular carriers for drug delivery and contrast imaging applications. Five commercially available polystyrene particles with different diameters (from 20 to 1000 nm) were used and imaged by using a 3dimensional confocal imaging techniques. The two proposed assays are: 'volumetric' assay which evaluate the spherical volume of internalized particles and 2) 'max-flat' assay which evaluate the total differential area between cells and internalized particles. These two assays were then compared to a reference method. Among these three assays, the 'max-flat' assay was found to be the most reliable and accurate to quantify and investigate the total content of internalized nanomaterials. The 'max-flat' assay also allowed for a 3dimensional subcellular investigation of the adaptation and localization mechanisms between cytoskeleton and internalized materials, which may help to further increase the selectivity and delivery of active and passive biopolymers. Therefore, we believe that the here presented assays could become a useful tool to address many biomaterials questions, especially where the key issue is the quantitative assessment of dose effect issues related to the size-dependence response of nanomaterials.

Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747(Received 1 September 2006)During immune responses, macrophages play important roles in phagocytosis and antigen presen-tation by engulfing pathogenic micro-organisms and cell debris. Since the function of a macrophagehighly depends on a series of physical steps including migration, direct contact and strong bindingto its target, deployment of cytoplasm and membrane, and intake of the target, here we have inves-tigated the mechanical behavior of a macrophage by manipulating it with a flexible pipette that wasused as a force sensor and transducer. We examined the response of a macrophage to mechanicalpulling by a positively charged pipette. We observed that the macrophage initially formed strongbinding to the pipette and migrated along the direction of pulling for some early period. Afterthat period, the macrophage exerted a huge traction force to pull the pipette back and attemptedto retract itself towards its original location. We found that whether it was able to return to theoriginal location depended on the level of applied force. Since the traction force generated by asingle macrophage had not been characterized accurately, we measured the force for the first timeto our knowledge and found the maximum traction force to be around 80 nN. This quantitativemeasurement was made possible by a new and convenient method used to calibrate the stiffness ofthe pipette. Through the study, we acquired a better understanding of the mechanics of and theforce generation by a macrophage.

Water extracts from meats and offals were incubated with a macrophage cell line (RAW 264.7), and nitrite in the medium was measured as an index of the macrophage stimulating activity. Ten of 38 water extracts had macrophage stimulants and chicken meat, chicken gizzard, cattle reticulorumen, swine stomach, and swine cerebrum had high activities.