Organocatalytic Diastereoselective (4 + 1) Cycloaddition of o-Hydroxyphenyl-Substituted Secondary Phosphine Oxides.

The first organocatalytic diastereoselective (4 + 1) cycloaddition of o-hydroxyphenyl-substituted secondary phosphine oxides (SPOs) has been established, which makes use of o-hydroxyphenyl substituted SPOs as suitable four-atom phosphorus-containing 1,4-dinucleophiles and 3-indolylformaldehydes as competent 1,1-dielectrophiles under BroÌ·nsted acid catalysis. The reaction mechanism was suggested to involve the formation of 3-indolylmethanol intermediates and vinyliminium intermediates, which played an important role in controlling the reactivity and diastereoselectivity of the (4 + 1) cycloaddition under BroÌ·nsted acid catalysis. By this approach, a series of benzo oxaphospholes bearing P- and C-stereocenters were synthesized in moderate to good yields (50%-95% yields) with excellent diastereoselectivities (all >95:5 dr). This reaction not only represents the first organocatalytic diastereoselective (4 + 1) cycloaddition of o-hydroxyphenyl-substituted SPOs but also provides an efficient and diastereoselective method for the construction of phosphorus-containing benzo five-membered heterocyclic skeletons bearing both P-stereocenter and C-stereocenter.

Immunomodulatory effects of Radix isatidis polysaccharides in vitro and in vivo.

Radix isatidis (R. isatidis) is a commonly used traditional Chinese herbal medicine, which has been used for thousands of years in China and is believed to have the pharmacological properties of heat-clearing and detoxification. Heat-clearing and detoxification are theories of traditional Chinese medicine meaning that R. isatidis could treat febrile disease by clearing heat and reducing swelling. Polysaccharides isolated from R. isatidis by water extraction and alcohol precipitation have exhibited numerous biological activities, including antiviral and immunomodulatory effects. The present study was performed to investigate the immunomodulatory effects of water-soluble R. isatidis polysaccharides (RIPs) on RAW264.7 macrophages and murine splenocytes, and attempt to preliminarily identify the mechanism of immunomodulation. In vitro, RIPs had a low cytotoxicity, as shown by CellTiter 96® AQueous One Solution Cell Proliferation Assay. RAW264.7 cells treated with different concentrations of RIP displayed different morphological changes, from a round shape and aggregation to polygonal shape and dispersion in a dose-dependent manner. In the 5 mg/ml RIP-treated group, the changes of morphology were as same as the lipopolysaccharide-treated group. RIP also significantly enhanced the release of nitric oxide as shown by Griess method, and the secretion of TNF-α and IL-6 in RAW264.7 cells was confirmed by ELISA assay. Western blotting revealed a significant increase of toll-like receptor-4 (TLR-4) in RIP-treated RAW264.7, suggesting that TLR-4 may be associated with the immunomodulatory mechanism of RIP. Animal experiments also demonstrated through ELISA assays a significant increase in IFN-γ and IL-10 levels after the splenocytes of RIP-immunized mice were stimulated by inactivated herpes simplex virus type 2. The immune function of RIP-immunized mice was improved. The present study suggested that RIP could be potentially used as a novel immunomodulator.

N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride: An immune-enhancing adjuvant for hepatitis E virus recombinant polypeptide vaccine in mice.

Adjuvants are essential for enhancing vaccine potency by improving the humoral and/or cell-mediated immune response to vaccine antigens. This study was performed to evaluate the immuno-enhancing characteristic of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC), the cationically modified chitosan, as an adjuvant for hepatitis E virus (HEV) recombinant polypeptide vaccine. Animal experiments showed that HTCC provides adjuvant activity when co-administered with HEV recombinant polypeptide vaccine by intramuscularly route. Vaccination using HTCC as an adjuvant was associated with increases of the serum HEV-specific IgG antibodies, splenocytes proliferation and the growths of CD4+CD8- T lymphocytes and IFN-γ-secreting T lymphocytes in peripheral blood. These findings suggested that HTCC had strong immuno-enhancing effect. Our findings are the first to demonstrate that HTCC is safe and effective in inducing a good antibody response and stimulating Th1-biased immune responses for HEV recombinant polypeptide vaccine.

Evaluation of Immunostimulatory Effects of N-(2-Hydroxy) Propyl-3-Trimethylammonium Chitosan Chloride for Improving Live Attenuated Hepatitis A Virus Vaccine Efficacy.

This study was to evaluate the immunostimulatory effects of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC) as an adjuvant for improving a commercial live attenuated hepatitis A virus (HAV) vaccine efficacy in mice. Mice in the experimental group were intraperitoneally immunized with a solution of HTCC and live attenuated HAV vaccine. And for those injected with sterile water, HTCC or live attenuated HAV vaccine were treated as mock group, negative group, and positive group in turn. The serum HAV-specific IgG titers and the ratios of the serum HAV-specific IgG2a/IgG1 in the experimental group were significantly increased (p = 0.00042 and p = 0.040, respectively). Splenocyte proliferation stimulation index in experimental group was higher than positive group (p = 0.021), and significantly higher than mock group and negative group (p = 0.0078 and p = 0.0050, respectively). The percentages of CD4+ T lymphocytes in the peripheral blood in experimental group were significantly higher than positive group, negative group, and mock group (p = 0.012, p = 0.012, and p = 0.045, respectively). Compared to the other three groups, experimental group showed a slightly higher ratio of CD4+/CD8+, but there were no significant differences (p > 0.05). In the percentages of CD8+ T lymphocytes, there were no significant differences among the four groups (p > 0.05). HTCC can enhance live attenuated HAV vaccine to generate stronger humoral responses and induce a Th1-biased immune response, as well as IgG2a class switching, compared with the live attenuated HAV vaccine alone. This study validated an important concept for further development of a safe and potent vaccine adjuvant.

A recombinant plasmid containing CpG motifs as a novel vaccine adjuvant for immune protection against herpes simplex virus 2.

The aim of the present study was to evaluate the efficacy of a herpes simplex virus type 2 (HSV-2) DNA vaccine co­immunized with a plasmid adjuvant containing CpG motifs. A novel eukaryotic expression plasmid vector containing kanamycin resistance gene (pcDNA3Kan) was acquired from pET­28a(+) and pcDNA3 plasmids. A gene encoding full length HSV­2 glycoprotein D (gD) was amplified from the pcDNA3­gD plasmid, which was cloned into pcDNA3Kan resulting in the construction of the recombinant plasmid pcDNA3Kan­gD (pgD). A DNA segment containing 8 CpG motifs was synthesized, and cloned into pcDNA3Kan, resulting in the recombinant plasmid pcDNA3Kan­CpG (pCpG). Mice were co­inoculated with pgD (used as a DNA vaccine) and pCpG (used as an adjuvant) by bilateral intramuscular injection. Mice inoculated with pgD+pCpG showed higher titers of antibodies than those inoculated with the DNA vaccine alone (P<0.05). In addition, mice inoculated with pgD+pCpG showed the highest percentage of CD4+ T cells in the blood of all the groups (P﹤0.05). Thus, the present study demonstrated that pCpG could stimulate the HSV­2 DNA vaccine to induce a stronger cell­mediated immune response than the DNA vaccine alone. The aim of the present study was to evaluate the efficacy of a HSV­2 DNA vaccine (pgD) co­immunized with a plasmid adjuvant containing CpG motifs (pCpG). Whether the pCpG would be able to stimulate the pgD to induce a stronger immune response compared with pgD alone.

Experimental infection of Z:ZCLA Mongolian gerbils with human hepatitis E virus.

AIM: To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus (HEV). METHODS: Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acute hepatitis E patient, and virus pathogenesis was assessed in this host. Non-infected gerbils served as the control group. Feces samples from gerbils were collected weekly for reverse transcription-nested polymerase chain reaction. Serum anti-HEV IgG and alanine aminotransferase (ALT) were detected by enzyme linked immunosorbent assay. At sacrifice, each animal's liver, spleen and kidney were collected for histopathologic examination. RESULTS: HEV-infected gerbils showed fatigue, with histopathological changes observed in the liver, spleen and kidney. HEV RNA was detected in fecal samples taken at day 7 after inoculation and the detectable levels lasted out to day 42 after inoculation. Interestingly, ALT levels were only moderately increased in the HEV-infected animals compared with the non-infected control group. CONCLUSION: Z:ZCLA Mongolian gerbils are susceptible to human HEV.

Purification and partial characterization of recombinant Cu, Zn containing superoxide dismutase of Cordyceps militaris in E.coli.

The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35+/-3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS-PAGE analysis and 94.2+/-1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80+/-2% residual activity at pH 7.8, at 50 degrees C for 4h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD).