Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels—against these antigens from subjects with O. viverrini-positive HBD—higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA > severe HBD > mild HBD > healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.     

Development and Optimization of Micro/Nanoporous Osmotic Pump Tablets

Micro/nanoporous osmotic pump tablets coated with cellulose acetate containing polyvinylpyrolidone (PVP) as pore formers were fabricated. Propranolol hydrochloride was used as a model drug in this study. Formulation optimization based on USP 31 requirements was conducted following a central composite design using a two-level factorial plan involving two membrane variables (pore former and coating levels). Effect of molecular weight of pore former (PVP K30 and PVP K90) was also evaluated. Responses of drug release to the variables were analyzed using statistical software (MINITAB 14). Scanning electron microscopy and atomic force microscopy showed that the pores formed by PVP. The drug release was dependent on the molecular weight and concentration of PVP and the level of coating. The results showed that acceptable 12-h profile could be achieved with only specific range of PVP K30-containing membrane at the defined membrane thickness. However, satisfactory 24-h profile could be accomplished by both PVP K30 and PVP K90-containing membrane at the range and membrane thickness tested. Preparation and testing of the optimized formulation showed a good correlation between predicted and observed values.     

α-Tocopherol and lipid profiles in plasma and the expression of α-tocopherol-related molecules in the liver of Opisthorchis viverrini-infected hamsters

Opisthorchis viverrini infection induces inflammation-mediated oxidative stress and liver injury, which may alter α-tocopherol and lipid metabolism. We investigated plasma α-tocopherol and lipid profiles in hamsters infected with O. viverrini. Levels of α-tocopherol, cholesterol, and low-density lipoprotein increased in the acute phase of infection. In the chronic phase, α-tocopherol decreased, while triglyceride and very low-density lipoprotein increased. Notably, high-density lipoprotein decreased both in the acute and chronic phases. In the liver, cholesteryl oleate, triolein, and oleic acid decreased in the acute phase, and increased in the chronic phase. Such chronological changes were negatively correlated with the plasma α-tocopherol level. The expression of α-tocopherol-related molecules, ATP-binding cassette transporter A1 (ABCA1) and α-tocopherol transfer protein, increased throughout the experiment. These results suggest that O. viverrini infection profoundly affects on lipid and α-tocopherol metabolism in due course of infection.     

Development and Evaluation of Chitosan-Coated Liposomes for Oral DNA Vaccine: The Improvement of Peyer’s Patch Targeting Using a Polyplex-Loaded Liposomes

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer’s patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 μg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation.     

Development of Push–Pull Osmotic Tablets Using Chitosan–Poly(Acrylic Acid) Interpolymer Complex as an Osmopolymer

The objectives of this study were to prepare push–pull osmotic tablets (PPOT) of felodipine using an interpolymer complex of chitosan (CS) and poly(acrylic acid) (PAA) as an osmopolymer, and to study the mechanisms of drug release from these tablets. The interpolymer complexes were prepared with different weight ratios of CS to PAA. Preparation of PPOT involved the fabrication of bilayered tablets with the drug layer, containing felodipine, polyethylene oxide, and the polymeric expansion layer, containing the CS–PAA complex. The effects of polymer ratios, type of plasticizers, and compression forces on release characteristics were investigated. It was found that drug release from PPOT exhibited zero-order kinetics and could be prolonged up to 12 or 24 h depending on the plasticizer used. PPOT using dibutyl sebacate showed a longer lag time and slower drug release than that using polyethylene glycol 400. In the case of polyethylene glycol 400, an increase in the CS proportion resulted in an increase in the drug release rate. The compression force had no effect on drug release from PPOT. Drug release was controlled by two consecutive mechanisms: an osmotic pump effect resulting in the extrusion of the drug layer from the tablet and subsequent erosion and dissolution of the extruded drug layer in the dissolution medium. The mathematical model (zero-order) related to extrusion and erosion rates for describing the mechanism of drug release showed a good correlation between predicted and observed values.     

Chitosan Nanoparticle Encapsulated Hemagglutinin-Split Influenza Virus Mucosal Vaccine

Subunit/split influenza vaccines are less reactogenic compared with the whole virus vaccines. However, their immunogenicity is relatively low and thus required proper adjuvant and/or delivery vehicle for immunogenicity enhancement. Influenza vaccines administered intramuscularly induce minimum, if any, mucosal immunity at the respiratory mucosa which is the prime site of the infection. In this study, chitosan (CS) nanoparticles were prepared by ionic cross-linking of the CS with sodium tripolyphosphate (TPP) at the CS/TPP ratio of 1:0.6 using 2 h mixing time. The CS/TPP nanoparticles were used as delivery vehicle of an intranasal influenza vaccine made of hemagglutinin (HA)-split influenza virus product. Innocuousness, immunogenicity, and protective efficacy of the CS/TPP-HA vaccine were tested in influenza mouse model in comparison with the antigen alone vaccine. The CS/TPP-HA nanoparticles had required characteristics including nano-sizes, positive charges, and high antigen encapsulation efficiency. Mice that received two doses of the CS/TPP-HA vaccine intranasally showed no adverse symptoms indicating the vaccine innocuousness. The animals developed higher systemic and mucosal antibody responses than vaccine made of the HA-split influenza virus alone. The CS/TPP-HA vaccine could induce also a cell-mediated immune response shown as high numbers of IFN-γ-secreting cells in spleens while the HA vaccine alone could not. Besides, the CS nanoparticle encapsulated HA-split vaccine reduced markedly the influenza morbidity and also conferred 100% protective rate to the vaccinated mice against lethal influenza virus challenge. Overall results indicated that the CS nanoparticles invented in this study is an effective and safe delivery vehicle/adjuvant for the influenza vaccine.     

Distribution and Abundance of Opisthorchis viverrini Metacercariae in Cyprinid Fish in Northeastern Thailand

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.