Optimal threshold of stimulated serum thyroglobulin level for 18F-FDG PET/CT imaging in patients with thyroid cancer.

This study was to explore the optimal threshold of thyroid-stimulating hormone (TSH)-stimulated serum thyroglobulin (s-Tg) for patients who were to receive 18F-fluorodeoxyglucose (18F-FDG) PET/CT scan owing to clinical suspicion of differentiated thyroid cancer (DTC) recurrence but negative post-therapeutic 131I whole-body scan (131I-WBS). A total of 60 qualified patients underwent PET/CT scanning from October 2010 to July 2014. The receiver operating characteristic (ROC) curve analyses showed that s-Tg levels over 49 µg/L led to the highest diagnostic accuracy of PET/CT to detect recurrence, with a sensitivity of 89.5% and a specificity of 90.9%. Besides, bivariate correlation analysis showed positive correlation between s-Tg levels and the maximum standardized uptake values (SUVmax) of 18F-FDG in patients with positive PET/CT scanning, suggesting a significant influence of TSH both on Tg release and uptake of 18F-FDG. So, positive PET/CT imaging is expected when patients have negative 131I-WBS but s-Tg levels over 49 µg/L.

Diagnostic value of Tg and TgAb for metastasis following ablation in patients with differentiated thyroid carcinoma coexistent with Hashimoto thyroiditis.

PURPOSE: This study was designed to investigate the clinical value of serum thyroglobulin (Tg) and antithyroglobulin antibody (TgAb) measurements and the cutoff value after ablation in differentiated thyroid carcinoma (DTC) complicated by Hashimoto thyroiditis (HT) with metastasis. MATERIALS AND METHODS: We measured serum Tg and TgAb levels and evaluated the disease status in 164 cases of DTC coexistent with HT in pathologically confirmed patients after surgery and post-remnant ablation during a 3-year follow-up. All Tg and TgAb levels were assessed by chemiluminescent immunoassay (IMA). Receiver operating characteristic (ROC) curve analysis was used to evaluate the prognostic value of Tg and TgAb for disease metastasis. The relationship between Tg and TgAb was analyzed using the scatter diagram distribution method. RESULTS: We found that the cutoff values of Tg and TgAb were 1.48 µg/L and 45 kIU/L, respectively. The area under the ROC curve (AUC) of Tg and TgAb was 0.907 and 0.650, respectively. CONCLUSIONS: In DTC coexistent with HT patients, the optimal cutoff value correlated with metastasis in Tg and TgAb was 1.48 µg/L and 45 kIU/L, respectively.

Structure-activity relationship of a guanine-free oligodeoxynucleotide as immunopotent inhibitor.

Excessive innate immune response could contribute to the pathogenesis of inflammatory and autoimmune diseases. It is required to develop agents to inhibit the overwhelming innate immune response. SAT05f, an inhibitory ODN with CCT repeat sequence found in human microsatellite DNA, has been demonstrated to down-regulate TLR7/9-mediated innate immune response, protect mice from D-GalN/CpG ODN induced lethal shock, and reduce anti-ssDNA antibody level in the lupus-prone mice induced by chronic graft versus host disease (cGVHD). In this article, to explore the structure-activity relationship of SAT05f, we designed and synthesized a series of ODNs based on the sequence of SAT05f by changing repeat number of the CCT unit, substituting CCT unit with AAG at 3' end or 5' end or in the middle and by forming hairpin at 5' or 3' end, and tested their inhibitory effect on the CpG ODN induced proliferation and TNF-α production in murine immune cells. The results indicated that 1) at least 8 CCT units were required for a CCT repeat ODN to display its inhibitory activity; 2) CCT unit at 3' end of SAT05f was necessary for its full inhibitory activity; and 3) 5' end of SAT05f could be modified to design a more potent SAT05f derived inhibitory ODN. The data provided here would be helpful for finding a potent inhibitory ODN as a candidate medicament for the treatment of diseases associated with over-activated innate immune response.

[Computer-aided-designing and eukaryotic expression of recombinant HPV 16 vcaccine].

AIM: To develop a recombinant protein vaccine with epitopes on L1 protein for prevention of HPV16 infection. METHODS: One B cell epitope and one T cell epitope from HPV16 L1 protein were chosen to construct genes encoding different recombinant proteins. Then all the 3D structures of the proteins were predicted by Geno3D in internet. An optimal protein named RH was chosen in which three B epitopes' location simulates that in the real HPV16 L1. Gene X (consisting of B+T+T) and gene Y (consisting of B+T) were constructed by multiple PCR. RH gene (consisting of X+X+X+X+Y+Y+Y) was constructed, sub-cloned into pFastbac1, and expressed in Sf9. RESULTS: RH protein with epitopes on HPV16 L1 was designed and predicted by Geno3D. RH gene was constructed and sub cloned into pFastbac1. Finally, it was successfully expressed in baculovirus expression system. CONCLUSION: A HPV16 recombinant vaccine is successfully constructed and expressed.

[Expression, purification and activity analysis of BCG HSP70.].

AIM: To get BCG HSP70 protein with excellent biologic activity through E.coli. expression and purification. METHODS: BCG HSP70 gene was amplified by PCR and inserted into vector pMD18-T. After confirmed by sequencing, the gene was subcloned into expression vector pET28a. Recombinant pET28a/HSP70 was transformed into E.coli. BL21(DE3). Recombinant BCG HSP70 protein was expressed with IPTG induction and the purified protein was then identified by SDS-PAGE and Western blot. And its effect on the proliferation of mouse splenocytes was observed. RESULTS: Gene encoding BCG HSP70 which was identical with that published in GenBank was successfully obtained by PCR. SDS-PAGE analysis showed a protein with relative molecular mass of 70 000 was expressed. When the purified protein was detected by Western blot analysis, a specific protein with a molecular mass of 70 000 could be visualized. The purity of the purified protein was about 96.5%. The purified protein could stimulate the proliferation of mouse splenocytes significantly. CONCLUSION: BCG HSP70 is expressed and purified successfully, which would lay a foundation for further research on BCG HSP70 and BCG.

[Establishment of a B16 cell line stably expressing genes encoding HER2 multi-epitope peptides in vivo].

AIM: To establish a B16 cell line stably expressing genes encoding HER2 multi-epitope peptides in vivo. METHODS: Eukaryotic expressing vector pcDNA3-GFP-HER2 was constructed by molecular cloning technique and transfected into B16 cells mediated by cationic liposome. After screened with G418, the transfected cells were passaged in vivo. The GFP-HER2 positive cells were isolated from tumor burdening mice by flow cytometry (FCM) sorting, and then monoclonized in the absence of G418 to obtain a B16 cell line stably expressing genes encoding HER2 multi-epitope peptides in vivo. This cell line was then identified after being passaged in vivo. RESULTS: Restriction endonulease analysis and DNA sequencing showed that the pcDNA3-GFP-HER2 was constructed and a B16 cell line stably expressing genes encoding HER2 multi-epitope peptides in vivo was obtained successfully. The GFP-HER2 positive proportion maintained higher than 90% after being passaged in vivo. CONCLUSION: A B16 cell line stably expressing genes encoding HER2 multi-epitope peptides is established successfully, which would provide a method to establish other cell lines stably expressing exogenous genes.

[Effect of a C-type CpG ODN on CVB3-infected mice].

AIM: To investigate the potential effect of a self-designed C-type CpG ODN (D-SL01) on coxsackie virus B3 (CVB3) infection. METHODS: HeLa cells infected by CVB3 were used as cell a model and BALB/c mice inoculated with CVB3 were used as an animal model for evaluating the anti-viral effect of D-SL01. RESULTS: The supernatants of D-SL01-treated PBMC protected HeLa cells from CVB3 infection and Vero E6 cells from VSV infection. However, in vivo study showed that D-SL01 reduced the body weight of CVB3-infected mice and aggravated the pathological changes related to myocarditis in mice when they were intraperitonealy injected for 6 days in succession. CONCLUSION: When used for the treatment of viral-infected diseases in vivo, CpG ODN may function in a complicated way, which indicates its administration time, route and dosage, should be taken into full consideration.

[Determination of trace copper (II) based on fluorescence quenching of NaGdF4 : Eu nanoparticles].

Water-soluble NaGdF4 : Eu fluorescent nanoparticles modified by citrate were synthesized by hydrothermal method with stable fluorescent properties. It was found that the fluorescence of the solution of as-prepared particles could be quenched by Cu2+, and thus a new mathod to determine trace Cu2+ using NaGdF4 : Eu as fluorescent probe was established. A pH 10.0 and the concentration 1.0 x 10(-3) mol x L(-1) of NaGdF4 : Eu were selected for measurement Besides, the effect of some foreign ions on the fluorescence signals was investigated and the interference of Fe3+ was found, which was eliminated by adding triethanolamine. The regression equation of standard curve was I = 532-0.685c with the correlation coefficient of -0.998 4 when the concentration of Cu2+ was in the range of 3.33 x 10(-6) -1.33 x 10(-4) mol x L(-1), and the detection limit of 8.9 x 10(-7) mol x L(-1) and a RSD of 0.62% for 11 replicates of a 6.0 x 10(-5) mol x L(-1) Cu2+ solution were obtained, which suggest a wide linear analytical range, high sensitivity and high precision. Analytical applicability of the particles was demonstrated by tea sample analysis and the results of Cu2+ determination were in good agreement with those obtained by atomic absorption spectrometry. The reason for fluorescence quenching by Cu2+ can be explained in terms of combination of Cu2+ with citrate on the surface of NaGdF4 : Eu particles leading to a change in surface structure and the composition.

[Determination of optimized MTT colorimetric assay as the identification method for the activity of B type CpG ODN BW006].

AIM: BW006, B type CpG ODN, will be approved as vaccine adjuvant for human use. So it is necessary to determine a stable, safety and easy-to-operate method for activity identification of BW006 with different lots. METHODS: According to the characteristic of BW006 to stimulate the proliferation of PBMC or mice splenocytes, MTT assay was preferentially selected to test the characteristic of BW006. The splenocytes of female BALB/c mice was stimulated by BW006, and different experiment parameters including splenocyte numbers, the shape of plate, BW006 concentration, culture medium, culture time and optimized MTT conditions were determined in these courses. RESULTS: The whole experiment scheme was defined as following: 6 x 10(5) splenocytes were co-cultured with 3 mg/L BW006 in 200 microliter RPMI1640(without phenol red) supplemented with 100 mL/L FBS in a 96 well square plate. PBS, the solvent of BW006 was used as negative control, and culture medium without cells was used as blank. After being co-cultured for 36 hours at 37 degrees C in humidified incubator with 50 mL/L CO(2), 100 microliter supernatants was aspired out and 10 microliter MTT (5 g/L) was added into each well. The plate was further incubated in dark at 37 degrees C for 4 hours that is sufficient for the formation of formazan. Afterward, 150 microliter DMSO was directly added into each well. The plate was shaken on plate shaker for nearly 20 minutes until the formazan was completely dissolved and detected A(578) value at at ELISA reader. CONCLUSION: Optimized MTT assay, which is non-radioactive contamination, low-price and simple operation, could be used as activity identification of BW006 during large-scale production.

[Establishment, optimization and application of a screening method for immunoregulatory oligodeoxynucleotides].

AIM: To establish a set of reasonable and convenient experimental system to provide a screening method for the development of novel immunoregulatory oligodeoxynucleotides. METHODS: The human PBMCs were stimulated by CpG ODN and/or immunoregulatory ODN. The cell proliferation and anti-viral activity of the supernatant induced by CpG ODN were examined by thymidine incorporation and anti-viral bioassay to evaluate the immunoregulatory activity of candidate ODN. The experimental conditions were also optimized. RESULTS: A screening method on which A151, a positive immunoregulatory ODN, inhibited the proliferation and anti-viral activity of the CpG ODN-induced human PBMCs was successfully established. CONCLUSION: The successful establishment of CpG ODN based screening method lays the foundations for further development of novel immunoregulatory oligodeoxynucleotides.

[Enhancement of HSP-MUC1 antitumor activity by type C CpG-ODN BW005].

AIM: To investigate the enhancement of anti-tumor effect of HSP-MUC1 by self-designed type C CpG-ODN BW005. METHODS: The immunostimulatory effect of CpG-ODN BW005 was detected by IFN protection assay and (3)H proliferation assay in vitro. Sixty C57BL/6 mice were separated into 5 groups randomly, including Sodium Chloride control, HSP-MUC1 control, HSP-MUC1/1585, HSP-MUC1/1826 and HSP-MUC1/BW005. Mice were injected s.c. with agents on day 0, 14 and 28 and were implanted MUC1-EL4 tumor cells s.c. on day 33. Tumor growth and murine death were recorded. Blood was collected in 57 day from tail vein. Subtype of anti-HSP and anti-MUC1 IgG in serum was detected by indirect ELISA. RESULTS: CpG-ODN BW005 could stimulate the proliferation of hPBMC and mice spleoncyte and IFNalpha production. HSP-MUC1/BW005 postponed tumor development, with the average tumor-developed day of 44.8, and prolonged the survival of mice with the average survival day of 49.5. Moreover, final tumor-developed rate of this group was 33.33%, which was the lowest; final survival rate of this group was 66.67%, which was the highest. Levels of anti-HSP and anti-MUC1 IgG2a in HSP-MUC1/CpG-ODNs group were enhanced. CONCLUSION: CpG-ODN BW005, a kind of type C CpG-ODN, could enhance the anti-tumor effect of HSP-MUC1.

[Expression of predicted B cell epitope peptide in S2 subunit of SARS coronavirus spike protein in E.coli and identification of its mimic antigenicity].

AIM: To study the expression of predicted B cell epitope peptide in S2 subunit of SARS coronavirus spike protein in E.coli and its mimic antigenicity to S2 protein. METHODS: B cell epitopes in S2 subunit of SARS coronavirus spike protein was predicted using DNAStar software. The cDNA sequence encoding the B cell epitope peptide was constructed artificially by PCR and then cloned into the downstream of chaperone 10 gene in vector pET28a(+) to construct pET28-chap10-S2epi plasmid. The fusion protein, chap10-S2epi, was expressed in E.coli BL21(DE3) and identified by SDS-PAGE and Western blot. The rabbit was immunized by purified Chap10-S2epi for the preparation of antiserum, which was used to identify the mimic antigenicity of Chap10-S2epi to S2 protein by ELISA. RESULTS: Chap10-S2epi fusion protein was successfully constructed and expressed in E.coli. The antiserum from the animal immunized by Chap10-S2epi recognized full length of SARS coronavirus S2 spike protein. CONCLUSION: The predicted B cell epitope peptide of SARS coronavirus S2 spike protein can induce the antigenicity of S2 protein, which provides some fundamental data for developing engineering vaccine against SARS coronavirus infection.

[The fusion construction of HIV-1 Tat gene and efficient expression in E.coli].

AIM: To express high-level the Tat protein in E.coli. METHODS: Full-length HIV-1 Tat gene was amplified artificially by PCR and Tat gene was mutated site-specifically (substitution the codons AAG encoding the lysine at the 28th and the 50th site by the CAG encoding glutamine) in order to eliminate the transcriptional activity of Tat protein. The site-mutated Tat gene was fused with chaperone10 gene, and then was subcloned into vector pET28a. The recombinant plasmid was expressed in E.coli BL21(DE3). The expressed products were identified by Western blot. RESULTS: Full-length HIV-1 Tat gene was amplified successfully by three rounds of PCR. The recombinant plasmid pET28a-chaperone 10-Tat was expressed efficiently in E.coli BL21(DE3). Western blot analysis showed the expressed Tat fusion protein with relative molecular mass (M(r)) 24 000 could bind to anti-His-tag monoclonal antibody. CONCLUSION: Full-length HIV-1 Tat gene was cloned and chaperone 10-Tat fusion protein was expressed efficiently in E.coli BL21(DE3), which will lay the foundation for researching the pathogenic effect of HIV-1 Tat on AIDS.

[Discovery of Cervus nippon Temminck-derived sentrin/SUMO].

In order to identify unknown encoding cDNAs of Cervus nioppon Temminck (sika deer), we constructed a cDNA library of uterus from Jilin-Shuangyang Cervus nippon Temminck using PCR cDNA library kit. PCR products of the library were cloned into pGEM-Teasy vectors and the cDNAs were sequenced and analyzed by nucleotide homology comparison against GenBank Database using the BLAST network service. The results showed that the cDNA library contained cDNA fragments of different lengths and a full length encoding cDNA highly homologous to human sentrin-1/SUMO-1 (small ubiquitin-related modifier 1) was identified. The cDNA was deposited in GenBank under the accession number AF 242526. These show that Cervus nippon Temminck-derived sentrin/SUMO gene has been discovered from PCR cDNA library of uterus from Cervus nippon Temminck.