Comparative Studies of Bioactivities and Chemical Components in Fresh and Black Garlics.

To investigate the bioactivities of fresh garlic and its processed product, black garlic, we conducted comparative analyses of antioxidant, anti-inflammatory, innate immune activation, and anti-cancer activities in addition to the chemical composition (sugar, amino acid, and polyphenol contents) of these materials. Simultaneous assay using neutrophil-like cells showed that fresh garlic exhibited antioxidant and innate immunostimulatory activities, whereas black garlic displayed a potent anti-inflammatory effect. The antioxidant activity index was correlated with phenol and flavonoid contents, while the innate immunostimulatory activity was correlated with fructan content. Furthermore, some black garlics with low fructose content were found to inhibit the proliferation of UM-UC-3 cancer cells, while other black garlics rich in fructose increased UM-UC-3 cell proliferation. It was shown that the processing of fresh garlic could change the composition of sugars, antioxidants, and amino acids, which have different effects on neutrophil-like cells and UM-UC-3 cells, as well as on bioactivities.

Effect of fat ingestion on postprandial oxidative status in healthy young women: a pilot study.

Reactive oxygen species (ROS) and highly reactive oxygen species (hROS) secreted by leukocytes are crucial to innate immunity; however, they pose a risk of oxidative stress. To monitor their balance in daily health check-ups, optical technologies for the simultaneous measurement of ROS (superoxide radicals) and hROS (hypochlorite ions) that utilize only a few microliters of whole blood have been developed. The aim of this study was to clarify whether this system could assess the effects of fat ingestion on postprandial oxidative status. Eight healthy young Japanese women ingested a beverage containing oral fat tolerance test cream. Blood samples were collected before and 0.5, 1, 2, 4, and 6 h after fat ingestion. Blood ROS and hROS levels, oxidative stress markers, and biochemical markers were monitored. Consistent with previous studies, triglyceride levels significantly increased at 4 h (p<0.01) and returned to near-baseline levels 6 h after ingestion. ROS levels peaked significantly at 2 h (p<0.05), and hROS levels peaked significantly at 1 (p<0.05) and 2 h (p<0.01) after ingestion. This study offers an insight into the acute effects of fat ingestion on leukocyte activity and provides a methodology for monitoring postprandial oxidative status.

Comparison of the oxidative profiles before and after revascularization in peripheral arterial disease: a pilot study.

Reactive and highly reactive oxygen species (ROS and hROS) produced by white blood cells are essential for innate immunity; however, they may cause oxidative stress in the host. We developed systems for simultaneously monitoring ROS and hROS, i.e., superoxide radicals (O2•-) and hypochlorite ions (OCl-) secreted from stimulated white blood cells in a few microliters of whole blood. We previously reported on the evaluation of healthy volunteers' blood using the developed system; however, whether patients' blood can be assessed remains unclear. Here, we report a pilot study of 30 cases (28 patients) with peripheral arterial disease, in whom we measured the ROS and hROS levels before and approximately one month after endovascular treatment (EVT) using the system (CFL-H2200) that we developed. At approximately the same time points, physiological indices of blood vessels, oxidative stress markers, and standard clinical parameters in the blood were also monitored. The ankle-brachial index, a diagnostic tool for peripheral arterial disease, was significantly improved after EVT (p<0.001). The ROS-hROS ratio, low-density lipoprotein cholesterol, and hematocrit levels were decreased after EVT (p<0.05), while triglyceride and lymphocyte levels were increased after EVT (p<0.05). The correlations between the study parameters were also analyzed.

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes.

Shikonin, an anti-inflammatory compound of "Shikon", inhibits the neutrophil superoxide (O2 (•-)) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca(2+) concentration ([Ca(2+)]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca(2+)]i (by fluorescence) and O2 (•-) generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca(2+)]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca(2+) release (IICR) and store-operated Ca(2+) entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca(2+)]i and O2 (•-) generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca(2+) fluxes showed that shikonin inhibits IICR and IICR-linked O2 (•-) generation (IC50 values: 0.28 and 0.31 µM for [Ca(2+)]i and O2 (•-), respectively), as well as SOCE and SOCE-linked O2 (•-) generation (IC50 values: 0.39 and 0.25 µM for [Ca(2+)]i and O2 (•-), respectively). These results suggested that shikonin inhibits the O2 (•-) generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca(2+) fluxes such as IICR and SOCE.

Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

The Effects of Adlay Tea Intake on Immune Homeostasis and Vascular Endothelial Function in Healthy Adults: A Randomized, Double-Blind, Parallel-Group Comparative Study.

Excessive immune response and inflammation are associated with an increased risk of various diseases. In particular, excessive myeloperoxidase (MPO) activity in neutrophils causes inflammatory reactions and lifestyle-related diseases. Adlay has a long history of being used as a traditional Chinese medicine. Polyphenols present in adlay seeds are expected to have the effect of suppressing excessive immune and inflammatory responses. Here, we conducted a randomized, double-blind, parallel group, placebo-controlled study was conducted to evaluate the suppressing effects of adlay seeds extract on excessive immune responses. One hundred and twenty adults participated in the study and they were equally divided into an adlay tea intake group and a placebo group. MPO activity was significantly elevated in the placebo group after 8-wk ingestion, while no significant change was observed in the adlay group. Vascular endothelial functions improved in the adlay group, especially in subjects over 40 y old. These results indicate that adlay tea intake may suppress an excessive immune and inflammatory responses, and improve arterial stiffness. Since caffeic acid, p-coumaric acid, and ferulic acid detected in adlay tea are known to inhibit MPO activity, these polyphenols may be the major functional molecules. Collectively, adlay tea is considered to have a preventative effect against lifestyle-related diseases through improving vascular endothelial function by effects to maintain immune homeostasis of the contained polyphenols. This trial was registered at University Hospital Medical Information Network Clinical Trials Registry (UMIN000032263).

Correlation between human health and reactive oxygen species produced in blood: a long-term chemiluminescence and fluorescence analysis.

The previous slide-glass type system could simultaneously detect reactive and highly reactive oxygen species, i.e., superoxide radicals (O2-·) and hypochlorite ions (OCl-) elicited from leucocytes in sample blood, but had some drawbacks, i.e., signal noise from air-flow stirring, potential biohazard risks, etc. because of open samples placed on a slide glass. We overcame these drawbacks by adopting a fluidic-chip container in a new system, which resulted in higher sensitivity and more stable measurements. Using the new system, we conducted a pilot study on nominally healthy volunteers to find whether or not the monitored activities of leukocytes can distinguish more or less unhealthy conditions from healthy ones. At first, healthy volunteers of both genders and of various ages showed that the fluctuation magnitudes (%) of O2-· and OCl- were nearly similar to each other and to that of the neutrophil count fluctuation. These parameters sometimes exceeded the healthy fluctuation range. By comparing these large fluctuations with the data of an inflammation marker C-reactive protein (CRP), the neutrophil count fluctuation and the timings/symptoms of abnormalities found in questionnaire, we could gain information suggesting the factors causing the large fluctuations. The new system could detect bodily abnormalities earlier than CRP or self-aware symptoms.

Utility of delayed fluorescence as endpoint for rapid estimation of effect concentration on the green alga Pseudokirchneriella subcapitata.

Algal growth inhibition tests for environmental risk assessment require improved efficiency to evaluate large numbers of chemicals. As an endpoint for rapid estimation of the effect concentration of test chemicals, we propose the delayed fluorescence (DF) measurement from an alga 24 h after exposure. Eight chemicals (bifenox, bromoxynil, bensulfuronmethyl, diuron, diflufenican, thiobencarb, m-chlorophenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) were tested. The EC50 values from the 24 h DF measurement were similar to those from the conventional 72 h growth test for seven tested chemicals excepting thiobencarb. We conclude that 24 h DF measurement is a possible endpoint for rapid estimation of the EC50 values obtained in the 72 h growth test for those seven chemicals.

New feature of delayed luminescence: preillumination-induced concavity and convexity in delayed luminescence decay curve in the green alga Pseudokirchneriella subcapitata.

A new method for measuring delayed luminescence (delayed fluorescence) employs preillumination and a dark waiting period before normal excitation. The preillumination results in a concavity and a convexity in the decay curve in delayed luminescence in the green alga Pseudokirchneriella subcapitata. Formation of the concavity and the convexity is not affected by excitation wavelength (680 nm and 700 nm). However, the concavity and the convexity progressively decrease as the dark waiting period increases after preillumination. The formation of the concavity and the convexity was inhibited by exposure to the electron transport inhibitors DBMIB (644 microg/L, 2.0 microM) and Antimycin A (55 microg/L, 0.1 microM). Samples exposed to DBMIB exhibited noticeable reduction in the concavity, whereas samples exposed to Antimycin A exhibited pronounced reduction in the convexity. There is a possibility that the formation and disappearance of the concavity and the convexity are due to the reduction-oxidation state of the plastoquinone pool and the cyclic electron transport. We expect this method being useful in evaluating the effects of chemicals (particularly toxic chemicals) on photosynthetic reactions, and the method may also help to resolve questions regarding the source of long delayed luminescence.

[Activation of human neutrophils by hemin].

Activation of neutrophils by free heme is considered as one of the mechanisms for cellular dysfunction under the conditions of hemorrhage or tissue damage. We studied about the effects of hemin, ferriprotoporphyrin IX, on human neutrophil activation by measurements of adhesion molecule expression and reactive oxygen species (ROS) production. Human neutrophils purified from heparinized blood of healthy volunteers were stimulated with hemin. Surface expression of CD11b and L-selectin were evaluated by flow cytometry, and superoxide production was detected by chemiluminescence. Hemin increased the expression of CD11b and produced superoxide accompanying by increase in intracellular free calcium concentration. Thus, free heme-molecule is suggested to possess the activity to initiate or aggravate tissue injuries. Since neutrophils do not express CD163, scavenger receptor for hemoglobin-haptoglobin complex, the mechanisms by which hemin exerts these effects are still to be studied.

Elucidating the Improvement in Vascular Endothelial Function from Sakurajima Daikon and Its Mechanism of Action: A Comparative Study with Raphanus sativus.

Vascular diseases, such as myocardial and cerebral infarctions, are the leading causes of death. Some vascular diseases occur as the result of decreases in vascular endothelial function. The innermost layer of the vasculature is formed by vascular endothelial cells (VECs), which are critical for nitric oxide (NO) synthesis. In our search for active constituents in farm products with the potential for improving the vascular system, we examined the effect of Raphanus sativus cv. Sakurajima Daikon on NO production in VECs. In this study, we found that the underlying mechanism for stimulating NO production by Sakurajima Daikon extract involves endothelial-NO-synthase (eNOS) activation by the phosphorylation of Ser1177 and the dephosphorylation of Thr495, which are triggered by elevated concentrations of cytoplasmic Ca2+ resulting from the activation of Ca2+ channels in VECs. We observed that trigonelline, an active constituent of Sakurajima Daikon, improves NO production in VEC cultures.

Rapid on-site dual optical system to measure specific reactive oxygen species (O2-• and OCl-) in a tiny droplet of whole blood.

Oxidative stress has been implicated in various disorders and controlling it would be important for healthy life. We have developed a new optical system for easily and accurately measuring oxidative stress in whole blood. It is optimized for simultaneously detecting reactive oxygen species (ROS) and highly reactive ROS (hROS), elicited mostly by white blood cells in a few microliters of blood. Results obtained by using this system show at least four important findings. 1) chemiluminescence of MCLA was confirmed to be attributable to O2-•. 2) PMA-stimulated cells released O2-• longer and more slowly than fMLP-stimulated ones. 3) fluorescence produced by APF oxidation was confirmed to be attributable to hROS, mostly OCl-, produced by myeloperoxidase. 4) the generation of OCl- was found to be a slower process than the O2-• generation. We also conducted pilot studies of oxidative stress in healthy volunteers.

Oral administration of Pantoea agglomerans-derived lipopolysaccharide prevents metabolic dysfunction and Alzheimer's disease-related memory loss in senescence-accelerated prone 8 (SAMP8) mice fed a high-fat diet.

The pathogenesis of Alzheimer's disease (AD) remains unclear, but an imbalance between the production and clearance of amyloid-ß (Aß) peptides is known to play a critical role in AD progression. A promising preventative approach is to enhance the normal Aß clearance activity of brain phagocytes such as microglia. In mice, the intraperitoneal injection of Toll-like receptor 4 agonist was shown to enhance Aß clearance and exhibit a preventative effect on AD-related pathology. Our previous clinical study demonstrated that orally administered Pantoea agglomerans-derived lipopolysaccharide (LPSp) exhibited an LDL (low-density lipoprotein)-lowering effect in human volunteers with hyperlipidemia, a known risk factor for AD. In vitro studies have shown that LPSp treatment increases Aß phagocytosis by microglial cells; however it is still unclear whether orally administered LPSp exhibits a preventive effect on AD progression. We show here that in senescence-accelerated prone 8 (SAMP8) mice fed a high-fat diet, oral administration of LPSp at 0.3 or 1 mg/kg body weight·day for 18 weeks significantly improved glucose metabolism and lipid profiles. The LPSp treatment also reduced pro-inflammatory cytokine expression and oxidative-burst activity in the peripheral blood. Moreover, LPSp significantly reduced brain Aß burden and memory impairment as seen in the water maze test, although we could not confirm a significant enhancement of Aß phagocytosis in microglia isolated from the brains after treatment. Taken together, our results show that LPSp holds promise as a preventative therapy for AD or AD-related diseases induced by impairment of metabolic functions.

Oral administration of Pantoea agglomerans-derived lipopolysaccharide prevents development of atherosclerosis in high-fat diet-fed apoE-deficient mice via ameliorating hyperlipidemia, pro-inflammatory mediators and oxidative responses.

Pantoea agglomerans (P. agglomerans) is a Gram-negative bacterium that grows symbiotically with various edible plants, and the oral or sublingual administration of lipopolysaccharide derived from P. agglomerans (LPSp) have been suggested to contribute to prevention of immune-related diseases. Our previous study indicated that orally administered LPSp was shown to exhibit an LDL-lowering effect in hyperlipidemic volunteers; however, a preventive effect of LPSp on atherosclerosis is unclear. The present study attempted to evaluate the anti-atherosclerotic effect by LPSp in a mouse model of high-fat diet (HFD)-induced atherosclerosis. For 16 weeks, apoE-deficient mice were fed an HFD and received drinking water containing LPSp (0.3 or 1 mg/kg body weight/day). The results showed that the orally administered LPSp decreased body weight. A significant reduction in atherosclerotic plaque deposition was observed even with the lower dose of LPSp. The biochemical analyses showed that LPSp markedly improved glucose tolerance and reduced plasma LDL and oxidized LDL levels. In addition, LPSp significantly reduced the production of pro-inflammatory mediators including MCP-1 (in the plasma), TNF-α and IL-6 (in the colon), and decreased the oxidative burst activities in the peripheral blood sample. Taken together, these results suggest the possibility that oral administration of LPSp can effectively ameliorate HFD-induced hyperlipidemia and inflammatory/oxidative responses to prevent atherosclerosis and related metabolic disorders.

Evaluation of a Hypertensive Rat Model Using Peripheral Blood Neutrophil Activity, Phagocytic Activity and Oxidized LDL Evaluation.

BACKGROUND/AIM: A system is being developed that can be used to easily evaluate the health condition of an individual with the help of trace amounts of a blood sample by focusing on xenobiotics. The system is called "Multimodal homeostasis evaluation system" (measurement of neutrophil activity, phagocytic activity of phagocytes and quantification of oxidized LDL (OxLDL)). To elucidate the possibility of using this system as an evaluation system for the health condition of an individual, clearly explaining the changes in various diseases is essential. In this study, evaluations were carried out using hypertensive model animals. MATERIALS AND METHODS: Spontaneously hypertensive model rats SHR/NCrlCrlj and control rats WKY/NCrlCrlj were raised for 10 weeks from 6 to 16 weeks of age and their blood pressure was measured over time. Blood neutrophil activity (superoxide anion (O2•-) generation and myeloperoxidase (MPO) activity) and phagocytic activity of phagocytes was measured by our developed apparatus (a simple prototype device under development). OxLDL was measured by an ELISA kit, and biochemical markers were measured using the blood sample. RESULTS: Compared to WKY rats of the control group, systolic blood pressure, diastolic blood pressure, and mean blood pressure of SHR rats increased significantly with age. In SHR rats, there was a significant elevation in O2•- generation and MPO activity of neutrophils, alanine aminotransferase and triglycerides of blood, while phagocytic activity, OxLDL, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol and total-bilirubin decreased. CONCLUSION: In the hypertensive model, biochemical markers were found to have a relationship with O2•- generation, MPO activity, phagocytic activity of phagocytes, and OxLDL. This system is expected to be useful for clinical monitoring of hypertension diseases.

Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

BACKGROUND/AIM: Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. MATERIALS AND METHODS: Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 µg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. RESULT: The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. CONCLUSION: The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult. These findings provide promising evidence that assessment of individual phagocytic capacity can be made easier using this novel device.

Cilostazol suppresses adhesion of human neutrophils to HUVECs stimulated by FMLP and its mechanisms.

The interaction between neutrophils and endothelial cells (ECs) is of great importance in many physiological and pathological progresses. Although cilostazol (CLZ), a novel selective phosphodiesterase (PDE) type 3 inhibitor, has been proved to be useful in vasodilatation and inhibition of platelet aggregation, its effect on adhesion is not clearly known. In this study, we examined the effects and investigated the mechanisms of cilostazol on neutrophil adhesion to human umbilical endothelial cells (HUVECs) triggered by N-formyl-methionyl-leucyl-phenylal-anine (FMLP), a chemotactic peptide. The soluble vascular cell adhesive molecule-1 (sVCAM-1) release from FMLP (10 microM)-stimulated HUVECs was determined by ELISA kits. Fluo-2, a fluorescent indicator, was used to investigate intracellular free calcium concentration ([Ca2+]i) in HUVECs. HL-60 cells were induced to be neutrophilic by DMSO and loaded with Fluo-3, another fluorescent indicator, to detect [Ca2+]i, and CLA was used as a chemiluminescent indicator to determine superoxide production in neutrophilic cells. The result showed that CLZ (1-100 microM) significantly inhibited neutrophil adhesion to FMLP-stimulated HUVECs. In HUVECs, CLZ obviously downregulated sVCAM-1 level, while it had no meaningful influence [Ca2)]i. But in neutrophils, FMLP-activated superoxide generation and [Ca2+]i increase were found being inhibited by exposure to CLZ . Furthermore, we also demonstrated that Ca2+ increase was preceded to the superoxide generation in neutrophils. The results suggest that CLZ involves in adhesion reactions between neutrophil and ECs, partly via VCAM-1 expression in ECs, and decreasing [Ca2+]i induced activation of neutrophils, which means a lot to prevent atherosclerosis and other cardiovascular diseases.

Rapid ecotoxicological bioassay using delayed fluorescence in the green alga Pseudokirchneriella subcapitata.

A rapid and simple ecotoxicological bioassay allows quick estimation of the effects of Simazine (CAT) or 3,5-dichlorophenol (3,5-DCP) on the growth of the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The effects of a 15-min exposure to CAT or 3,5-DCP on delayed fluorescence (DF) in P. subcapitata were compared to the results of standard growth inhibition tests involving 72h of exposure to these chemicals at the same concentrations. Integrated DF intensity in the time period from 0.6 to 50s was found to correlate with algal growth inhibition as measured by the standard tests. In addition, the behaviour of DF in this time period exhibited characteristic kinetics indicative of the chemical being tested.

Development of an Evaluation Device for Phagocytic Activity of New Phagocytes Using Simple and pH-sensitive Particles that Do Not Require Pre-treatment.

BACKGROUND/AIM: Phagocytic activity is affected by a number of different stress and age-dependent factors. An easy measurement of phagocytic activity is thought to allow an indicator of an individual's health. In this study, we investigated conditions of measurement to easily evaluate the activity of phagocytosis of phagocytic cells (macrophages and neutrophils) using an easy-to-use prototype, which was improved from the device by Hamamatsu Photonics K.K., to detect neutrophil activity using subtle fluorescence. MATERIALS AND METHODS: pH-sensitive fluorescent particles (pHrodo-Green E. coli Bio particles, GE particles) were added to mouse-derived macrophage cell lines (J774.1) and then incubated for 2 h at 37°C. For negative control, the phagocytosis inhibitor cytochalasin D (CyD), was added prior to culture. Next, fluorescence intensity was measured by the Prototype to evaluate the phagocytic activity of macrophages and neutrophils. Phagocytosis was also confirmed by flow cytometry. RESULTS: The Prototype detected a steady fluorescence increase in 5 sec in J774.1 after phagocytosis, using GE particles as a negative control in the presence of CyD. Furthermore, detection was possible at 10(4) cells/test, a concentration where the flow cytometer had difficulty for detection. CONCLUSION: The Prototype enables measurement of the phagocytic activity within a short period of time, even with a small sample amount, thus establishing the basic conditions of measurements of phagocytosis.

Simultaneous monitoring of superoxides and intracellular calcium ions in neutrophils by chemiluminescence and fluorescence: evaluation of action mechanisms of bioactive compounds in foods.

We have developed a measuring system for simultaneous monitoring of chemiluminescence and fluorescence, which indicate respectively, (i) generation of superoxide anion radicals (O2(-•)) and (ii) change in the intracellular calcium ion concentration ([Ca(2+)]i) of neutrophils triggered by the mechanism of innate immune response. We applied this measuring system for establishing a method to distinguish between anti-inflammatory actions and antioxidant actions caused by bioactive compounds. We evaluated anti-inflammatory agents (zinc ion [Zn(2+)] and ibuprofen) and antioxidants (superoxide dismutase [SOD] and ascorbic acid). It was shown that ibuprofen and Zn(2+) were anti-inflammatory while SOD and ascorbic acid were anti-oxidative. We conclude that it is possible to determine the mechanism of action of bioactive compounds using this method.