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Background Honokiol has been reported to possess anti-inflammatory and neuroprotective activities. However, the poor aqueous solubility of honokiol limits its clinical application for systemic administration. Purpose This study aims to develop a novel formulation of nanosome-encapsulated honokiol (NHNK) for intravenous therapy against mouse experimental autoimmune encephalomyelitis (EAE) that mimics human multiple sclerosis. Methods Nanosomes and NHNK were prepared by using an ultra-high pressure homogenization (UHPH) method. Mice were treated with NHNK or empty nanosomes during the peak phase of EAE symptoms. Symptoms of EAE were monitored and samples of the spinal cord were obtained for histopathological examinations. Results The stock of NHNK containing honokiol in the nanosome formulation, which showed the structure of single phospholipid bilayer membranes, was well formulated with the particle size of 48.0 ± 0.1 nm and the encapsulation efficiency 58.1 ± 4.2%. Intravenous administration of NHNK ameliorated the severity of EAE accompanied by a significant reduction of demyelination and inflammation in the spinal cord. Furthermore, NHNK decreased the number of IL-6+, Iba-1+TNF +, Iba-1+IL-12 p40+, and CD3+IFN-γ+ cells infiltrating the spinal cord. Conclusion The UHPH method simplified the preparation of NHNK with uniformly distributed nanosize and high encapsulation efficiency. Intravenous administration of NHNK ameliorated the severity of EAE by suppressing the infiltration of activated microglia and Th1 cells into the spinal cord. Collectively, these results suggest that the formulation of NHNK is a prospective therapeutic approach for inflammatory CNS diseases, such as multiple sclerosis.

High plasma triglyceride (TG) concentration in fasting state could cause hemorheological abnormality, thus increasing the incidence of metabolic diseases. Exercise has been reported to effectively reduce postprandial TG response. This study aimed to investigate whether a single bout of pre-prandial exercise can affect lipemia and hemorheological variables after a high-fat meal. Nine healthy young male subjects completed two experimental trials. The subjects walked for 1 h at 50% maximal oxygen uptake (V̇O₂max) (the exercise, EX trial), or rested (the control, CON trial). In the next morning, the subjects consumed a high-fat meal, and the postprandial lipemia and hemorheological responses were monitored for 6 h. The results showed that postprandial plasma TG concentrations were significantly lower in the EX trial compared to the CON trial. The postprandial low-density lipoproteins (LDL) concentration declined in the first 2 h and then gradually returned to the baseline level in both trials. The postprandial blood viscosity also decreased in the CON trial. There was no significant difference in postprandial blood viscosity, red blood cell (RBC) deformation index and aggregation degree between the trials. There was no significant correlation between plasma TG concentration and blood viscosity. In conclusion, brisk walking effectively reduced postprandial TG concentration, but has no significant impact on postprandial blood viscosity, RBC deformation index and RBC aggregation index.

The C57BL/6J mice were fed a 135-day normal diet or a high-fat diet (HFD) without, or concurrent with, a single yam dioscorin (80 mg/kg) or dipeptide NW (40 mg/kg) intervention every day. The final body weights (g) of mice were 26.1 ± 1.4, 34.97 ± 2.1, 31.75 ± 2.6, and 31.66 ± 3.1, respectively, for normal diet-fed, HFD-fed, dioscorin-intervened, and NW-intervened group. The mice in both intervened groups showed similar less weight gains and had significant differences (P0.05) compared to those in the HFD group under the same cumulative HFD intakes. The blood biochemical index of mice with dioscorin interventions showed significantly lower contents in total cholesterol and low-density lipoprotein, and NW interventions showed significantly lower total triglyceride contents compared to those of the HFD group (P0.05). Both intervened mice exhibited similar reductions in total visceral lipid contents and have significant differences compared to those of the HFD group (P0.05). The dioscorin intervention was better than NW interventions in lowering blood glucose levels by oral glucose tolerance tests and both showed significant differences (P0.05) compared to those in the HFD group. Yam dioscorin or dipeptide NW will potentially be used for preventive functional foods of less body weight gains and impaired glucose tolerance controls, which require further clinical trial investigations.

Cisplatin is a potent anti-cancer drug, however, its accompanied organ-toxicity hampers its clinical applications. Cisplatin-associated kidney injury is known to result from its accumulation in the renal tubule with excessive generation of reactive oxygen species. In this study, we encapsulated honokiol, a natural lipophilic polyphenol constituent extracted from Magnolia officinalis into nano-sized liposomes (nanosome honokiol) and examined the in vivo countering effects on cisplatin-induced renal injury. We observed that 5 mg/kg body weight. nanosome honokiol was the lowest effective dosage to efficiently restore renal functions of cisplatin-treated animals. The improvement is likely due the maintenance of cellular localization of cytochrome c and thus preserves mitochondria integrity and their redox activity, which as a consequence, reduced cellular oxidative stress and caspase 3-associated apoptosis. These improvements at the cellular level are later reflected on the observed reduction of kidney inflammation and fibrosis. In agreement with our earlier in vitro study showing protective effects of honokiol on kidney cell lines, we demonstrated further in the current study, that nanosuspension-formulated honokiol provides protective effects against cisplatin-induced chronic kidney damages in vivo. Our findings not only benefit cisplatin-receiving patients with reduced renal side effects, but also provide potential alternative and synergic solutions to improve clinical safety and efficacy of cisplatin treatment on cancer patients.

Vaccination is the most effective means of preventing infectious diseases; however, few vaccines are effective against Streptococcus iniae (S. iniae) in grouper. This work presents an efficacious and safe vaccine against S. iniae infections in the grouper Epinephelus coioides. The vaccine candidate was the S. iniae GSI-310 strain. The vaccination was administered by intraperitoneal injection, and consisted of formalin-inactivated antigens combined with an AS-F or ISA763A adjuvant. Peripheral blood samples were collected for RT-qPCR and phagocytosis and agglutination assays. Our results indicated that immunoglobulin M (igm) was maximally expressed in the two vaccinated groups at 3 months post-secondary vaccination (PSV). A significant upregulation of mRNA expression for interleukin-1β (il-1β) and tumor necrosis factor-α (tnf-α) was also observed in fish treated with antigens combined with ISA763A, which peaked at 3 months PSV. In fish treated with antigens combined with AS-F, il-1β and tnf-α expression peaked at 14 days post-primary vaccination (PPV). Phagocytic activity and index increased significantly in the two vaccinated groups. Furthermore, fish in the two vaccinated groups exhibited significantly elevated agglutination titers compared to fish in the control group, in which almost no agglutination reaction was detected. In the efficacy test, the vaccinated and control groupers were treated with S. iniae at 1, 3, and 6 months PSV. The relative percentage survival (RPS) values of antigens with AS-F and antigens with ISA763A were both 100% at 1 and 3 months PSV; at 6 months PSV, the RPS values for these groups were 100% and 97.7%, respectively. Furthermore, the level of protection observed in the field trial closely resembled that achieved on a laboratory scale. Therefore, the proposed vaccine mixed with AS-F or ISA763A improved immune responses and provided safe and long-lasting protection in farmed groupers.

The oral administration of Asn-Trp (NW) or carnosine (β-alanyl-L-histidine) dipeptides to D-galactose (Gal)-induced BALB/c mice was used to evaluate antioxidant activities in vivo. D-Galactose (Gal) was subcutaneously injected into the dorsal necks of mice daily for eight weeks to induce oxidative stress (Gal group). From the beginning of the fifth week, groups of NW10, NW40 (10 or 40 mg NW kg(-1)) or carnosine40 (40 mg carnosine kg(-1)) were administered orally concurrent Gal injection until the end of studies. It was found that the malondialdehyde (MDA) contents in these intervention groups were much lower than the Gal group. The mice in the NW40 group showed significant improvements compared to the Gal group in a reference memory task and probe trial test evaluated by Morris water maze. Mice in the intervention groups showed higher GSH levels and oxygen radical antioxidant capacity activities and lower MDA levels in the brain or liver tissues compared to the Gal group. The levels of advanced glycation end-products, including N(ε)-(carboxymethyl)lysine (CML) and argpyrimidine, in the brain tissues of the NW40 interventions are significantly lower compared to the Gal group. These results suggest that NW may be useful in developing functional foods for antioxidant and anti-aging purposes.

J Oral Pathol Med (2011) 40: 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre-cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid-derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen-stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)-sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol-enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b+Gr-1+ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL-10, arginase-I and iNOS in splenic CD11b+ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr-1+IL-10+ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid-derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca-related oral diseases.

The adjuvant effect of liposomes formulated with three phospholipids including phosphatidylcholine-liposomes (PC-Lip), phosphatidylserine-liposomes (PS-Lip), and stearylamine-liposomes (SA-Lip) was compared with virus alone using inactivated Newcastle disease virus (NDV) as a model antigen. The difference in adjuvanticity was evaluated using the haemagglutination-inhibition (HI) test, enzyme-linked immunosorbent assay, and a challenge study following intranasal inoculation of specific pathogen-free chickens. After two inoculations, a liposomal vaccine consisting of NDV in PC-Lip resulted in a significant increase in HI titre, up to 32-fold higher than a vaccine containing virus alone and 320-fold higher than a vaccine containing NDV in SA-Lip. PC-Lip also elicited a significant mucosal secretary immunoglobulin A response (P0.05) in tracheal lavages and a serum IgG response (P0.05). In response to viral challenge, all control animals died, whereas 90% of animals which received PC-Lip survived. The results suggest that PC-Lip may be suitable as an adjuvant for mucosal vaccination against NDV in chickens.

High dietary intake of fats has been thought to be one of the major risk factors for the development of CVD. Less is known about the possible influence of fats from various sources on haemorheological abnormalities, which are considered an important factor in the pathogenesis of these diseases. The goal of the present study was to investigate effects of high-fat diets enriched in unsaturated fatty acids (USFA), SFA ortrans-fatty acids (TFA), respectively, on haemorheological parameters in rats. Wistar female rats were divided into four groups and fed diets based on the AIN-93M formulation containing approximately 10 % energy from soyabean oil (control group) or 40 % energy from soyabean oil (USFA), palm oil (SFA) and vegetable shortening (TFA) for 8 weeks. The results showed that rats fed high-fat diets exhibited significant increases in serum TAG levels (P P P P P P P trans-fat and high-fat diets on haemorheological parameters.

A novel method for monitoring the growth of coliforms in milk was developed based on measuring the current change in an amperometric sensor. The sensor consists of a circuit with a homemade potentiostat and a pair of electrodes. The electrode was immersed in milk samples containing methylene blue with various concentrations of bacterial inoculums. The microbial metabolism led to the reduction of methylene blue resulting in a change of current. The time required to identify readily detectable change (detection time, DT) provided an approximate measurement of the amount of microorganisms in the initial inoculums. The sensor system used in this study has the selectivity towards coliform bacteria such as Escherichia coli and Enterobacter aerogenes. The calibration curve of DT against concentration of coliform showed a linear correlation coefficient (R2 = 0.9192) over the range of 102–108 CFU/mL. The sensor was able to detect the coliform bacteria at initial concentrations of 105 CFU/mL within 6 h, making it suitable for use in real-time monitoring of bacterial growth. This system has potential application in the detection of coliform concentration in milk at dairy farms when a proper selective media is designed.

BACKGROUND: It was reported in a previous paper that the yam tuber storage protein dioscorin exhibited antihypertensive effects on spontaneously hypertensive rats. The aim of the present study was to evaluate and compare the effects of packets of instant food (30 g) with (treated meal) and without (placebo) lyophilised yam powder on hypertensive subjects. RESULTS: A placebo-controlled feeding trial was conducted daily for 5 weeks (stage 1), followed by a 1 week washout and then a 5 week crossover (stage 2). Twenty-one subjects finished the trial. One packet of treated meal contained 140 ± 2.54 mg of dioscorin according to enzyme-linked immunosorbent assay. The blood pressure results of the treated meal and placebo groups at stage 1 end versus originals, but not at stage 2 end versus stage 2 beginning, were significantly different by the paired t test. Systolic (SBP) and diastolic (DBP) blood pressure readings after treated meal intervention, but not after placebo intervention, differed significantly from the original values based on one-way analysis of variance followed by the post hoc Tukey test; the reductions in SBP and DBP were 6.52 and 4.76 mmHg respectively. The feeding trial did not appear to affect serum lipid profiles or other biochemical measurements of cardiovascular risk. CONCLUSION: Intake of an instant food containing 140 mg of dioscorin over 5 weeks had a regulating effect on human blood pressure. Copyright © 2008 Society of Chemical Industry

Several reports have demonstrated that cantharidin is a strong anticancer compound in vitro; however, its in vivo usefulness is often limited due to its high systemic toxicity. In this study, we encapsulated cantharidin into pegylated liposomes and studied its activity against human breast cancer MCF-7 cells in vitro and its systemic toxicity in mice. Another two methods were also used to reduce the dosage of cantharidin, including labeling liposomal cantharidin with octreotide and exposing cells to hyperbaric oxygen. The cytotoxic activity of pegylated liposomal cantharidin was drastically reduced compared with free cantharidin in vitro. Octreotide-labeled pegylated liposomal cantharidin induced cell death by specifically targeting somatostatin receptors in MCF-7 cells. Cell death was augmented with a low dose of cantharidin under hyperbaric oxygen. Liposomal cantharidin had significantly less systemic toxicity than free cantharidin in vivo and also exhibited a high efficacy against antitumor growth in nude mice. These results suggest that the systemic toxicity of cantharidin can be mitigated by liposome encapsulation; however, that did not decrease its antitumor activity.

We studied the effects of multi- and single-target liposomal drugs on human gastric cancer cell AGS both in vitro and in vivo. The cytotoxic effect of dihydrotanshinone I was significantly enhanced by treatment with octreotide-polyethylene glycol(PEG)-liposome, Arg-Gly-Asp(RGD)-PEG-liposome, and RGD/octreotide-PEG-liposome encapsulated with 0.5 mug/ml of dihydrotanshinone I to AGS cell for 24 h, compared to control. Furthermore, the AGS cell survival rate for multi-target versus single target liposomal drugs was significantly suppressed. Microscopic examination revealed that significant cell death occurred in the multi- and single-target liposomal encapsulated drug groups. Significant suppression of tumor growth in AGS cell xenograft nude mice given octreotide-PEG-liposome, RGD/octreotide-PEG-liposome encapsulated drug, versus those given a free drug was noted after 13 d of experimentation with the multi-targeted liposome: up to 60.75% and 41.2% reduction of tumor volume as compared to dimethylsulfoxide (DMSO) control and the free drug groups respectively. The treated animals showed no gross signs of toxicity. The results have potential clinical application.

Taiwanofungus camphoratus (syn. Antrodia camphorata), a medicinal mushroom in Taiwan, is reputed to provide several therapeutic benefits, but the wild fruiting body is very rare. In this study, we used Taiwanofungus camphoratus extracts from wild fruiting bodies and two types of artificial cultivation (solid-state culture and liquid-state fermentation) to examine their anti-inflammatory effects in microglia cells and their possible roles in protection against neurodegenerative diseases. First, EOC13.31 microglia was treated with various kinds of Taiwanofungus camphoratus extracts and lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) to evaluate the iNOS expression. Western blot and RT-PCR analysis showed that among the various kinds of extracts from wild fruiting bodies, methanol extracts were the most potent inhibitors of iNOS expression. Secondly, the potency of methanol extracts could be ranked as follows: extracts of wild fruiting body>solid-state culture>liquid-state fermentation. To clarify the mechanisms involved, methanol extracts from fruiting body were found to inhibit the phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal protein kinases (JNK) and signal transducer and activator of transcription-1 (STAT-1) induced by LPS/IFN-gamma. Methanol extracts from fruiting body also inhibited NF-kappaB activation through the prevention of inhibitor kappaB (IkappaB) degradation. Moreover, methanol extracts from wild fruiting body inhibited both the iNOS and cyclooxygenase-2 (COX-2) expression induced by beta-amyloid in microglia in a dose-dependent manner. In an animal model, we confirmed that methanol extracts from fruiting bodies were able to suppress ear edema, indicating that they have anti-inflammatory activity in vivo. These results suggest that Taiwanofungus camphoratus exhibits an anti-inflammatory activity that might contribute to the prevention of neurodegenerative diseases.

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI(3)K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P(3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI(3)K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P(3) production and iNOS expression in LPS-activated macrophages. Inhibition of PI(3)K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI(3)K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-kappaB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI(3)K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.

Salvia miltiorrhiza Bunge, known as Danshen in Chinese traditional medicine is effective at promoting blood circulation and removing (or decreasing) blood stasis. In the present study, we selected aging, 24-month-old guinea pigs as the animal experimental models and fed them a diet containing 75, 100 or 150 mg/(kg day) of water-soluble extract components of Salvia miltiorrhiza Bunge (WSm) for 28 days, respectively, in order to evaluate the effects of WSm on their abnormal hemorheological parameters. The results showed that the blood biochemical parameters of the aging guinea pigs remained unaffected by orally given WSm compared to the controls, except that the fibrinogen levels of the group fed the high dose of WSm (150 mg/(kg day)) decreased. Aging guinea pigs fed a low dose of WSm (75 mg/(kg day)) showed no significant difference in hemorheological parameters. However, feeding of WSm at 100 mg/(kg day) (medium dose), significantly reduced erythrocyte membrane MDA levels, which probably increased erythrocyte deformability and decreased erythrocyte flow resistance, though no improvement in erythrocyte aggregation, blood viscosity, and blood viscoelasticity could be observed. Furthermore, when the dose reached 150 mg/(kg day) of WSm (high dose), a significant decrease in whole blood viscosity was observed at high, medium and low shear rates. Blood viscosity and viscoelasticity exhibited significant improvement in oscillatory measurements. Also, we found that the oxygen transport efficiency of whole blood increased.

The commercial polysaccharides of alginic acid (medium (3500 cps, 2% solution) and low (250 cps, 2% solution) viscosities) were esterified with acidic methanol (1 mmol L−1 HCl) at 4 °C with gentle stirring for 5 days to obtain methyl esters of medium-viscosity alginic acid (ME-MVA) and low-viscosity alginic acid (ME-LVA). These ME-MVA and ME-LVA were reacted with alkaline hydroxylamine to obtain medium-viscosity alginic acid hydroxamates (MVA-NHOH) and LVA-NHOH. The percentages of hydroxamic acid content in MVA-NHOH and LVA-NHOH were calculated as 25% and 20%, respectively. The hydroxamate derivatives of alginic acid were used to test the antioxidant and semicarbazide-sensitive amine oxidase (SSAO) inhibitory activities in comparison with original materials (MVA and LVA). The half-inhibition concentrations, IC50, of scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) were 24.5 and 29.8 µg mL−1 for MVA-NHOH and LVA-NHOH, respectively. However, few scavenging activities of the MVA and LVA were found at the same concentrations. The IC50 of the positive control of butylated hydroxytoluene was 5 µg mL−1. The scavenging activity of DPPH radical was pH-dependent, and the optimal pH for both of MVA-NHOH and LVA-NHOH was the Tris-HCl buffer (pH 7.9). Using electron spin resonance (ESR) to detect the activity of scavenging hydroxyl radicals, both alginic acid hydroxamates showed dose-dependent scavenging activities, and the IC50 was 90 and 92 µg mL−1, respectively, for MVA-NHOH and LVA-NHOH. Both alginic acid hydroxamates also exhibited protection against hydroxyl radical-mediated DNA damage. Both MVA-NHOH and LVA-NHOH showed dose-dependent inhibitory activities against bovine SSAO (2.53 units); the IC50 was 0.16 and 0.09 µg mL−1, respectively, for MVA-NHOH and LVA-NHOH, compared with 3.81 µg mL−1 of semicarbazide (positive controls). Amine oxidase activity staining also revealed that both MVA-NHOH and LVA-NHOH exhibited SSAO inhibitory activities. Both MVA-NHOH and LVA-NHOH showed mixed non-competitive inhibition against bovine SSAO. It was found that the V′max value was reduced and the K′m value was either increased (added MVA-NHOH, 0.05 µg mL−1) or reduced (added LVA-NHOH, 0.11 µg mL−1) in the presence of alginic acid hydroxamate. Copyright © 2006 Society of Chemical Industry

Background We reported that yam dioscorin and its peptic hydrolysates exhibited ACE inhibition and antihypertensive effects on SHRs, however, the active peptides are not really isolated until now. Using ACE inhibitory screenings, two penta-peptides, KTCGY and KRIHF, were selected for ex vivo and in vivo experiments. Results KTCGY, KRIHF, and captopril were shown to have similar vasodilating effects against phenylephrine (PE)-induced tensions in rat endothelium-dependent thoracic aortic rings, however, KTCGYKTCGY (two-repeated KTCGY) and TCGYTCGY (two-repeated TCGY) were showed endothelium-independent vasodilating effects against PE-induced tensions. KTCGY, KRIHF (10 or 20 mg/kg), and captopril (10 mg/kg) were used to evaluate antihypertensive activity during 24-h after a single oral administration to spontaneously hypertensive rats (SHRs). The KTCGY and KRIHF showed significantly different and reduced the systolic blood pressure of SHRs compared to the blank. Conclusions These results suggest that KTCGY and KRIHF may contribute important roles in yam dioscorin for regulating blood pressure in vivo.

Surface tension, foamability, and foam stability kinetics have been measured for the pure proteins bovine serum albumin (BSA) and beta-lactoglobulin, before and after aqueous solutions of the proteins had been subjected to different drying conditions, and also for whey protein concentrate (WPC). Pure proteins were air-dried, at 78 or 88 degrees C, in the presence and absence of sucrose or trehalose, at a mass ratio of 5:1 sugar/protein. WPC was spray-dried in the presence of various sugars: trehalose, sucrose, lactose, and lactitol. Spray-drying WPC without sugars resulted in a dramatic decrease in the foam stability, whereas drying in the presence of sugars gave better retention of the original foaming properties. Trehalose in particular resulted in almost complete retention of the foam stability observed for the nondried WPC. Pure beta-lactoglobulin showed similar behavior, but trehalose did not seem to afford the same protection to BSA.

Iridovirus is one of the most devastating viral pathogens in groupers (Epinephelus spp). The viral infection could often lead to near 100% mortality in hatcheries, thus a bottleneck to the development of the grouper aquaculture industry. Vaccination is an important and efficient strategy against viral infection. The most common delivery method for commercially available fish vaccines is injection, which is both time-consuming and labor intensive despite the high efficacy. We report here the development of a new adjuvant NE and its application in an immersion GIV subunit vaccine. The adjuvant NE can form low viscosity nanoparticles with average size of about 168 nm and are safe to grouper. When co-delivered with NE, green fluorescent protein (GFP) can be detected in gills and the digestive tract at 20 min postimmersion. Subsequently, an immersion subunit vaccine was generated by combining NE with recombinant major capsid protein (MCP) of GIV. After vaccination by immersion, MCP-specific antibodies could be detected at 2 weeks post-vaccination. Furthermore, the subunit vaccine enhanced the survival rate of infected fish in a dose-dependent manner. We further evaluated the vaccination protocol of this subunit vaccine. The results showed that boosters (at 1 or 2 weeks after the initial vaccination) enhanced the yield of specific antibodies and the protection against GIV in the vaccinated grouper at both 3 and 4 weeks after initial vaccination. Booster at 1 week had a greater impact than booster at 2 week did. At 10 weeks after initial vaccination, the fish were resistant to GIV in all groups; however, the viral loads in the booster groups was lower than those in the single-vaccination and control groups. In conclusion, out study indicates the potential application of this immersion subunit GIV vaccine as an effective and easy-to-handle vaccine for the grouper aquaculture industry.

Chien-Chang Shen,1,* Hong-Jen Liang,2,* Chia-Chi Wang,3 Mei-Hsiu Liao,4 Tong-Rong Jan11Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, 2Innovation and Incubation Center, Yuanpei University, Hsinchu, 3School of Pharmacy, Kaohsiung Medical University, Kaohsiung, 4Division of Isotope Application, Institute of Energy Research, Taoyuan, Taiwan*These authors contributed equally to this workBackground: It was recently reported that iron oxide nanoparticles attenuated antigen-specific humoral responses and T cell cytokine expression in ovalbumin-sensitized mice. It is presently unclear whether iron oxide nanoparticles influence T helper 1 cell-mediated immunity. The present study aimed to investigate the effect of iron oxide nanoparticles on delayed-type hypersensitivity (DTH), whose pathophysiology requires the participation of T helper 1 cells and macrophages.Methods: DTH was elicited by a subcutaneous challenge with ovalbumin to the footpads of mice sensitized with ovalbumin. Iron oxide nanoparticles (0.2–10 mg iron/kg) were administered intravenously 1 hour prior to ovalbumin sensitization. Local inflammatory responses were examined by footpad swelling and histological analysis. The expression of cytokines by splenocytes was measured by enzyme-linked immunosorbent assay.Results: Administration of iron oxide nanoparticles, in a dose-dependent fashion, significantly attenuated inflammatory reactions associated with DTH, including the footpad swelling, the infiltration of T cells and macrophages, and the expression of interferon-γ, interleukin-6, and tumor necrosis factor-α in the inflammatory site. Iron oxide nanoparticles also demonstrated a suppressive effect on ovalbumin-stimulated production of interferon-γ by splenocytes and the phagocytic activity of splenic CD11b+ cells.Conclusion: These results demonstrated that a single dose of iron oxide nanoparticles attenuated DTH reactions by suppressing the infiltration and functional activity of T helper 1 cells and macrophages in response to antigen stimulation.Keywords: iron oxide nanoparticle, T cell, macrophage, delayed-type hypersensitivity

Chien-Chang Shen1, Hong-Jen Liang2, Chia-Chi Wang3, Mei-Hsiu Liao4, Tong-Rong Jan1 1Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, 2Innovation and Incubation Center, Yuanpei University, Hsinchu, 3School of Pharmacy, Kaohsiung Medical University, Kaohsiung, 4Division of Isotope Application, Institute of Energy Research, Taoyuan, Taiwan Background: Accumulating evidence indicates that iron oxide nanoparticles modulate immune responses, and induce oxidative stress in macrophages. It was recently reported that iron oxide nanoparticles attenuated antigen-specific immunity in vivo, though the underlying mechanism remains elusive. The present study investigates the direct effect of iron oxide nanoparticles on antigen-specific cytokine expression by T cells, and potential underlying mechanisms. Methods: Ovalbumin-primed splenocytes were exposed to iron oxide nanoparticles, followed by restimulation with ovalbumin. Cell viability, cytokine production, and cellular levels of glutathione and reactive oxygen species were measured. Results: The splenocyte viability and the production of interleukin-2 and interleukin-4 were unaffected, whereas interferon-γ production was markedly attenuated by iron oxide nanoparticles (10–100 µg iron/mL) in a concentration-dependent manner. Iron oxide nanoparticles also transiently diminished the intracellular level of glutathione, with a peak response at 6 hours posttreatment. The effects of iron oxide nanoparticles on interferon-γ and glutathione were attenuated by the presence of N-acetyl-L-cysteine, a precursor of glutathione. However, iron oxide nanoparticles did not influence the generation of reactive oxygen species. Conclusion: Iron oxide nanoparticles induced a differential effect on antigen-specific cytokine expression by T cells, in which the T helper 1 cytokine IFN-γ was sensitive, whereas the T helper 2 cytokine interleukin-4 was refractory. In addition, the suppressive effect of iron oxide nanoparticles on interferon-γ was closely associated with the diminishment of glutathione. Keywords: iron oxide nanoparticle, T cell, antigen-specific, glutathione, cytokine

Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre-cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid-derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen-stimulated mice, we hypothesized that ANE might enhance the development of MDSC.Ovalbumin (OVA)-sensitized BALB/c mice were daily administered with ANE (5-50 mg/kg), polyphenol-enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses.ANE and PANE treatment significantly increased the spleen index and the population of CD11b(+) Gr-1(+) cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL-10, arginase-I and iNOS in splenic CD11b(+) cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr-1(+) IL-10(+) cells in the footpads challenged with OVA.Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid-derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca-related oral diseases.

Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and Angelica pubescens and has been used to treat several diseases, including metabolic syndromes. To investigate the hypoglycemic effects of osthole in diabetic db/db mice and the underlying mechanisms of these effects by in vitro assay, diabetic db/db mice and cell experiments were utilized to understand its possible effects. Osthole significantly activated both PPARalpha and PPARgamma in a dose-dependent manner based on the results of the transition transfection assay. The activation of PPARalpha and PPARgamma by osthole also resulted in an increase in the expression of PPAR target genes such as PPAR itself, adipose fatty acid-binding protein 2, acyl-CoA synthetases, and carnitine palmitoyltransferase-1A. In vitro results suggested that osthole might be a dual PPARalpha/gamma activator, but its chemical structure differed from that of the thiazolidinedione class of antidiabetic drugs. In addition, osthole markedly activated the AMP-activated protein kinase and its downstream acetyl CoA carboxylase molecules by increasing their phosphorylation levels. Finally, obese diabetic db/db mice were treated with osthole by different administered routes, and osthole was found to markedly reduce blood glucose level. Interestingly, osthole did not reduce the blood insulin or lipid levels, two phenomena that did occur in animals treated with insulin sensitizers like PPAR agonists. These results suggest that osthole can alleviate hyperglycemia and could be potentially developed into a novel drug for treatment of diabetes mellitus.

The aim of the present study was to elucidate the underlying mechanisms responsible for hemorheological abnormalities in elderly patients with Alzheimer's disease (AD).Twenty-one patients with AD and twenty-three age-matched healthy controls (CON) were studied. We used a controlled-shear rate rheometer generating various flow fields in vitro for simulating blood flow in vivo. The applied experimental techniques provided valid and quantitative data for the analysis of hemorheological abnormalities associated with AD. Principal blood biochemical parameters and hemorheological parameters, including blood viscosity, erythrocyte deformability, erythrocyte aggregation and oxygen transport efficiency of blood were assessed.The results show no statistically significant difference in most of the blood biochemical parameters between the AD patients and the CON, except that fibrinogen concentration and mean corpuscular cell volume level of erythrocytes (MCV) were significantly higher in the AD patients. Hemorheological parameters including blood viscosity, plasma viscosity, and blood viscoelasticity in the AD patients were considerably higher than the respective factors in the CON. Owing to the MDA levels of the AD patients being significantly higher than that of the CON, the AD patients also showed a decrease in erythrocyte deformability and an increase in blood flow resistance despite the lack of any significant difference in erythrocyte rigidity. In addition, the erythrocyte aggregation of AD patients was higher than that of the CON and reduced oxygen transport efficiency of blood was observed in the AD patients.The hemorheological abnormalities found in AD patients may be explained by the parallel findings of oxidative damage on erythrocyte membranes that could result in a decrease of erythrocyte deformability. Furthermore, the oxidative stress-induced elevation of fibrinogen concentration could lead to accelerated erythrocyte aggregation as a consequence of a rise in blood viscosity and blood viscoelasticity. Taken together, these factors may impair the oxygen transport efficiency of blood in AD patients.

BACKGROUND Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant of all the green tea catechins. The objective of the present study was to find out whether it improved the age-induced hemorheological abnormalities or not. METHODS AND RESULTS Twenty-four-month-old aging guinea pigs were used to test the effects of EGCG on hemorheological properties. Orally feeding EGCG at 30 mg x kg(-1) x day (-1) for 28 days resulted in a decrease in erythrocyte membrane malondialdehyde, and further improved erythrocyte deformability and blood viscosity at high and middle shear rates. In addition, it also significantly reduced erythrocyte aggregation, and improved blood viscosity at low shear rates and viscoelasticity at oscillatory flow. Consequently, efficiency of blood oxygen transport in aged guinea pigs increased after administration with EGCG. CONCLUSIONS Orally feeding EGCG 30 mg x kg(-1) x day(-1) for 28 days significantly improves the abnormal hemorheological parameters. These results suggest that EGCG has considerable potential as a substantial component for the development of new drugs or functional foods in improving the age-induced hemorheological abnormalities.

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

Background Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS). Methods Primary murine microglial cells were pretreated with IONPs (1–50 μg Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α was quantified by ELISA. The activity of IL-1β converting enzyme (ICE) and TNF-α converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively. Results Confocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1β, but not TNF-α. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE. Conclusions Our results demonstrated a contrasting effect of IONPs on the production of IL-1β and TNF-α by LPS-stimulated microglia, in which the attenuation of IL-1β by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing.