Shuhan Sun

Effects of a single nucleotide polymorphism on the expression of human tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)‐α plays a prominent role in inflammations and is a proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. Recent association studies have found that the TNF‐α−857T allele was associated with several disorders. Here we demonstrate, with reporter genes under the control of the two allelic TNF‐α promoters, that the minor allele −857T is a much stronger transcriptional activator than the major allele −857C in RAW264.7 cell line in response to lipopolysaccharide stimulation. However, the result was not consistent in HeLa cell line. Furthermore, for the quantitative analysis of TNF‐α synthesis between the −857C/C genotype from healthy subjects and the −857C/T genotype from AS patients, the quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay were performed separately. There was no significant difference between the two groups at the level of mRNA and protein. These results show that this polymorphism may have a direct effect on TNF‐α regulation in a tissue‐specific manner, and apart from the polymorphism at −857 in the TNF‐α promoter, there may be other factors affecting the expression of TNF‐α.

Epitope DNA Vaccines Against Tuberculosis: Spacers and Ubiquitin Modulates Cellular Immune Responses Elicited by Epitope DNA Vaccine

Cell‐mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3‐M‐38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38u2003kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala‐Ala‐Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3‐M‐S(spacer)‐38, p3‐Ub (UbGR)‐M‐S‐38 and p3‐Ub‐M‐38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H‐2b) mice. The cytotoxicity of T cells was detected by LDH‐release method and by enzyme‐linked immunospot assay for epitope‐specific cells secreting interferon‐γ. The results showed that DNA immunization with p3‐M‐38 vaccine could induce epitope‐specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein‐targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C‐terminus and protein‐targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi‐epitope DNA vaccines against tuberculosis.

Immune response in mice inoculated with plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus

This paper focuses on the development of candidate DNA vaccine encoding antigenic epitopes of type O foot-and-mouth disease virus (FMDV). A series of plasmids encoding different combinations of B cell epitopes and a T cell epitope were constructed and characterized by inoculating BALB/c mice. The specific antibodies were only detectable in the mice inoculated with plasmids encoding the T cell epitope and B cell epitopes from sites 5 and 1, within which site 5 includes residues 135-167 of VP1 and site 1 includes 141-160 region (G-H loop) and carboxyl terminus of VP1. Stronger cellular immune responses were also observed in these mice using T cell proliferation assay.

Effect of formaldehyde treatment on the recovery of cell-free fetal DNA from maternal plasma at different processing times.

BACKGROUNDnThe effect of formaldehyde treatment on the recovery of fetal DNA from maternal plasma is controversial. We evaluated the effect of formaldehyde and investigate the underlying mechanism.nnnMETHODSnBlood samples from pregnant women were treated or not treated with formaldehyde, and processed at different times. Total and fetal DNA in plasma was quantified by real-time polymerase chain reaction. Death and lysis of blood cells were assayed by trypan blue exclusion test. Plasma DNase activity was determined by the radial enzyme-diffusion method.nnnRESULTSnFormaldehyde addition showed no effect on the percentage of fetal DNA in samples processed 6 h after blood collection. In samples processed at 36 h, formaldehyde addition inhibited blood cell lysis and nuclease-mediated DNA degradation, thus markedly decreasing the concentration of total DNA and increasing the recovery of fetal DNA. The median (interquartile range) percentage of fetal DNA increased from 4.6% (3.8-6.8%) to 13.1% (10.3-17.0%).nnnCONCLUSIONnThe effect of formaldehyde on the percentage of fetal DNA in maternal plasma depends on processing time and is associated with prevention of cell lysis and inhibition of plasma DNase activity.

Enhanced Immunogenicity of Modified Hepatitis B Virus Core Particle Fused with Multiepitopes of Foot-and-Mouth Disease Virus

Abstract Hepatitis B virus core (HBc) particles, self‐assemble into capsid particles and are extremely immunogenic, hold promise as an immune‐enhancing vaccine carrier for heterologous antigens. However, formation of virus‐like particles (VLP) can be restricted by size and structure of heterlogous antigens. In the study, we investigated formation of VLP by modified HBc fused with specified foot‐and‐mouth disease virus (FMDV) multiepitopes and evaluated their immune effects. Firstly, three HBc display vectors (pHBc1, pHBc2 and pHBc3) were constructed by deletions of different lengths within the HBc c/e1 region: 75–78 amino acid (aa), 75–80 aa and 75–82 aa respectively. Secondly, we inserted different compositions of FMDV multiepitopes, BT [VP1(141–160)–VP4(21–40)] and BTB [VP1(141–160)–VP4(21–40)–VP1(141–160)], into modified regions. As a result, only plasmid pHBc3‐BTB of six recombinant vectors was expressed as soluble protein, which resulted in the formation of complete VLP confirmed by electron microscopy. Recombinant VLP could be taken up by cells and presented in vitro and in vivo. Furthermore, the modified VLP displayed a significantly stronger immunogenicity than other five recombinant proteins and GST‐BTB with a higher titer of peptide‐specific and virus‐specific antibody, elevated IFN‐γ and interleukin‐4 production, especially enhanced lymphocyte proliferation. The results encourage further work towards the development of FMDV vaccines using hepatitis B virus core particles fused with FMDV epitopes.

The binding of CpG-oligodeoxynucleotides to cell-surface and its immunostimulatory activity are modulated by extracellular acidic pH

Both the binding of CpG-oligodeoxynucleotides (CpG-ODNs) to cell-surface and its immunostimulatory activity were modulated by extracellular pH in present study. At neutral pH (pH 7.4), the binding of CpG-ODN to splenocyte-surface, as well as that of non-CpG-ODN, was competitively inhibited by non-specific DNA-Herring sperm DNA in a dose dependent manner, indicating their binding sites have no specificity for CpG-motif. When the extracellular pH shifted to acidic (pH 6.4), however, their binding to cell-surface markedly increased, and only the binding of non-CpG-ODN instead of CpG-ODN was inhibited by Herring sperm DNA, implying such pH change enabled CpG-ODN bind to its specific binding-site. Consistently, lymphocytes appeared more sensitive to the stimulation of CpG-ODN at acidic pH, and Herring sperm DNA inhibited the CpG-ODN-induced TNF production from splenocytes at pH 7.4, but not at pH 6.4. These results suggest the existence of membrane receptor that specifically engages CpG-ODN with high affinity only at acidic pH, and support the hypothesis that the binding CpG-ODN to its specific membrane receptor and subsequently triggering of CpG-related signaling occurred within acidified endosomes.

DNA Vaccination Using Bacillus Calmette‐Guerin‐DNA as an Adjuvant to Enhance Immune Response to Three Kinds of Swine Diseases

In order to enhance the immune efficacy of DNA vaccination, experiments were conducted to investigate the regulating effects of Bacillus Calmette‐Guerin (BCG)‐DNA as an adjuvant on immune responses of mice against foot‐and‐mouth disease (FMD), Aujeszkys disease (AjD) and classical swine fever (CSF). BCG‐DNA was purified from BCG by ion‐exchange chromatography. Three DNA vaccines (pVSG, pVgD and pVE2) against the respective infection were constructed, and BCG‐DNA was coimmunized to mice by muscle injection. The results showed that titres of specific immunoglobulin (Ig)G to the vaccines mounted remarkably in the sera of the adjuvant covaccinated mice (Pu2003<u20030.01). Antibody isotype IgG2a and IgG1 also increased, respectively, in mice coimmunized with BCG‐DNA compared with those of the control groups (Pu2003<u20030.01). Cellular immune cytokine interferon‐γ and cytotoxic T lymphocytes were detected in coimmunized BCG‐DNA groups (Pu2003<u20030.05). Whereas interleukin‐4, humoral immune cytokine, was not significant (Pu2003>u20030.05). These results suggest that codelivery of BCG‐DNA with DNA vaccines against FMD, AjD and CSF can enhance the induction of antigen‐specific, especially, cell‐mediated immunity.

Active Immunization with Glucose‐Dependent Insulinotropic Polypeptide Vaccine Influences Brain Function and Behaviour in Rats

Glucose‐dependent insulinotropic polypeptide (GIP) is involved in the aetiology of obesity induced by overnutrition, and blocking GIP activity may be valuable to anti‐obesity treatment. However, GIP and GIP receptor are closely related to various brain functions which have caused very little data to be published concerning this cerebral functionality after blocking GIP activity. Here, we showed that active vaccination of mature rats with GIP immunoconjugates [GIP‐keyhole limpet haemocyanin (KLH)] was associated with changes in body weight. Furthermore, we also observed significant changes in brain function and behaviour. Data indicated that GIP‐KLH‐immunized rats showed decreased spontaneous activity in the open field test, decreased cerebral glucose utilization assessed by 18F‐fluorodeoxyglucose‐positron emission tomography/computed tomography (PET/CT), and increased apoptosis and proliferation of hippocampal granule cells marked by the terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling (TUNEL) or proliferating cell nuclear antigen method. In conclusion, we have shown that vaccine‐induced antibodies inhibited GIP activity in vivo and led to significant changes in brain function and behaviour, which underscore the need to address any potential problems GIP‐targeted immunotherapy may involve in further research.

Association of HLA genes with ankylosing spondylitis in Han population of eastern China.

Ankylosing spondylitis (AS) is a chronic inflammatory disorder with a multifactorial genetic basis. HLA‐B27 was reported with the greatest susceptibility to AS but did not act alone. The aim of this study was to search for other gene(s) associated with AS independently of HLA‐B27 using 13 microsatellite markers spanning 1.5u2003Mb from locus TAP1 to HLA‐Cw and a single‐nucleotide polymorphism marker within NFκBIL1 gene promoter. Genotyping for microsatellites was performed in 175 AS patients of eastern Chinese and 219 ethnically matched healthy controls using polymerase chain reaction with fluorescence‐labelled primers, whereas the SNP marker was genotyped by direct DNA sequencing. Allele as well as haplotype frequencies were compared between cases and controls, and a linkage disequilibrium analysis was performed to estimate the LD relationship between the candidate regions. The frequencies of alleles D6S2811*128, STR_MICA*A5.1 and D6S2672*109, as well as haplotypes D6S2811*128–D6S2927*213–D6S2810*340, D6S2927* 221–D6S2810*350–MICA*A5.1, and D6S2810*350–MICA*A5.1–D6S2800* 136 were significantly increased in B27‐positive AS patients when compared with B27‐positive controls. The results indicated that there may be other gene(s) within the HLA region, especially around locus HLA‐B or HLA‐Cw, with susceptibility to AS independently of HLA‐B27.

Adjuvant Effects of Bacillus Calmette–Guerin DNA or CpG-oligonucleotide in the Immune Response to Taenia solium Cysticercosis Vaccine in Porcine

The immune stimulation properties of CpG‐oligonucleotides (CpG‐ODN) containing a central unmethylated CpG motif could be useful for vaccination against parasite infection. However, the high cost of synthetic CpG‐ODN has limited its use in veterinary vaccines. In this study, we investigated whether genomic DNA derived from Mycobacterium bovis bacillus Calmette–Guerin (BCG‐DNA) could be used as an effective adjuvant to enhance the immunogenicity and the protective capacity of recombinant cC1 antigen (rcC1) against pig cysticercosis. Pigs were vaccinated with rcC1 plus CpG‐containing DNA adjuvants (BCG‐DNA or CpG‐ODN) or rcC1 alone. Immunization with rcC1 alone induced a Th1‐biased response, whereas coadministration of rcC1 with BCG‐DNA or CpG‐ODN increased levels of IgG2, IFN‐γ, percentage of CD8+ and specific proliferation of peripheral blood mononuclear cells. Four weeks after the last immunization, pigs were infected with Taenia solium eggs. A high level of protection (81%) was induced by rcC1 immunization that was not significantly increased by the CpG‐containing DNA. These data indicate that coadministration of rcC1 plus BCG‐DNA or CpG‐ODN significantly enhanced Th1 response but did not improve the level of the protection induced.