Objective:This study evaluates the performance of chemiluminescence assay, which is designed to detect Hepatitis D Virus (HDV) Immunoglobulin G (IgG) antibodies.Methods:A comparative analysis was conducted among chemiluminescence anti-HDV IgG reagent, the magnetic particle-based domestic reagent A and domestic reagent B, and the Robo Gene HDV RNA kit, using 1909 HBsAg-positive plasma samples. This comparison aimed to delineate clinical specificity and detection accuracy. The anti-HDV IgG reagent precision was assessed at three different concentration levels following the Clinical Laboratory Standards Institute EP5-A2 guidelines. The specificity of the assay was validated using 200 HAV IgM positive, 545 HBsAg-positive but anti-HDV IgG-negative, 350 anti HCV positive plasma samples and 200 healthy human blood samples. Additionally, a concordance study was conducted with 545 HBsAg-positive and 37 anti-HDV IgG-positive plasma samples, comparing the anti-HDV IgG reagent against reagent A.Results:1 909 HBsAg-positive plasma samples were tested using 3 anti HDV IgG reagent and 1 HDV RNA reagent, 19 samples were identified as anti-HDV IgG-positive. The anti-HDV IgG demonstrated superior accuracy and specificity. The assay exhibited excellent precision, with intra-assay coefficient of variation (CV) values ranging from 1.57% to 4.30%, and inter-assay CV values between 1.71% and 4.67% for detecting samples at high, medium, and low concentration levels. Concordance with Reagent A showed consistent results in both positive and negative detections.Conclusion:In this study, the anti-HDV IgG reagent (chemiluminescence method) displayed outstanding specificity in detecting clinical samples and exhibited a high conformity rate with commercialized reagents, making it potentially suitable for screening anti-HDV IgG in HBsAg-positive samples.

Objective:This study aims to evaluate the quality and explore the preliminary clinical applications of a domestically developed hepatitis D virus nucleic acid quantification reagent (abbreviated as"domestic HDV RNA reagent").Methods:The sensitivity and accuracy of the reagent were evaluated in accordance with the WHO HDV RNA international standard, employing the Bio-Rad CFX Opus 96 real-time fluorescence quantitative PCR analysis system. Serial dilutions of pseudo-viruses or cell culture-derived virus were used to determine the linear range of the domestic HDV RNA reagent. Specificity was assessed using positive samples of HAV, HBV, HCV infection, and HEV national reference materials. Precision was evaluated with samples at both high and low concentrations. In a comparative analysis, 30 HDV IgG positive samples were tested using both the domestic HDV RNA reagent and the RoboGene HDV RNA kit based on the ABI 7500 FAST DX system. The Pearson correlation coefficient (r) was used to examine the correlation between the two reagents.Results:The domestic HDV RNA reagent demonstrated a high sensitivity of up to 6 IU/ml, consistent with that of the comparator reagent. The calibration curve for WHO HDV RNA standards had a slope of -3.286, with an amplification efficiency of 101.6%. The linear detection range spanned from 10 to 10 8 IU/ml for eight HDV genotypes. The domestic HDV RNA reagent exhibited exceptional specificity, without cross-reactivity observed with HAV, HBV, HCV, or HEV. Accuracy assessments at five concentration levels met the required standards, with intra-assay precision coefficient of variation ( CV) ranging from 1.20% to 4.20%, and inter-assay precision CV from 1.20% to 7.90%. The detection results for HDV IgG positive samples were highly correlated with the comparator reagent ( r=0.984, P<0.001), achieving a diagnostic accuracy of 100% compared to sequencing results. Conclusion:In this study, the domestic HDV RNA reagent possesses excellent specificity, accuracy, precision, and a broad linear range, attaining a sensitivity level on par with international reagents of the same type.

Objective:To propose a deep learning network model 2D-PE-GAN to automatically delineate the target area of nasopharyngeal carcinoma and improve the efficiency of target area delineation.Methods:The model adopted the architecture of generative adversarial networks which used a UNet similar structure as the generator, and 2D-PE-block was added after each layer of convolution operation of the generator to improve the accuracy of delineation. The experimental data included CT images from 130 cases of nasopharyngeal carcinoma. The images were preprocessed before model training. In addition, three models of UNet, GAN, and GAN with an attention mechanism were compared, and Dice similarity coefficient, Hausdorff distance, accuracy, Matthews correlation coefficient, Jaccard distance were employed to evaluate network performance.Results:Compared with UNet, GAN and GAN with the attention mechanism, the average Dice similarity coefficient of 2D-PE-GAN network segmentation of CTV was increased by 26%, 4% and 2%. The average Dice similarity coefficient of GTV segmentation was increased by 21%, 4%, 2%, respectively. Compared with the GAN network with the attention mechanism, the parameters and time of 2D-PE-GAN were reduced by 0.16% and 18%, respectively.Conclusions:Compared with the above three networks, 2D-PE-GAN network can increase the segmentation accuracy of nasopharyngeal carcinoma target area delineation. At the same time, compared with the attention mechanism with similar reasons, 2D-PE-GAN network can reduce the occupation of computing resources when the segmentation accuracy is not much different.

Objective:To evaluate the effect of upregulation of HuR gene on the radiosensitivity of esophageal squamous cell carcinoma cell Kyse450. Methods:The HuR gene of Kyse450 cells was upregulated by lentivirus. At the same time, X-ray irradiation at a dose of 6 Gy was selected as the intervention condition. Western blot and qPCR were used to detect the expression levels of protein and RNA after Kyse450 transfection, respectively. CCK8 kit was employed to determine the cell proliferation rate. Clone formation assay was adopted to evaluate the ability of cell clone formation. Wound healing experiment and the Transwell test were performed to detect changes in cell migration. Results:CCK8 assay showed that the proliferation ability of cells was enhanced after upregulation of HuR gene, and this enhancement trend was more obvious after radiation. The plate cloning experiment showed that with the increase of radiation dose, the clone formation rates were decreased in both groups, but the clone formation rate in the overexpression group was higher than that in the control group. Wound healing experiment and Transwell test demonstrated that the wound healing rate and migration ability in the overexpression group were higher than those in the control group, and the difference was more significant after radiotherapy. Western blot showed that the levels of MMP9 and MMP2 at 24 h after radiotherapy in the overexpression group were higher than those in the control group. Conclusion:The upregulation of HuR can enhance the proliferation, cloning, migration capabilities and decrease the radiosensitivity of esophageal squamous cell carcinoma cells.

Objective To investigate the significance of computed tomography(CT)and 3.0 T magnetic resonance imaging(MRI)in intensity-modulated radiotherapy(IMRT)for esophageal carcinoma. Methods Thirty-five patients newly diagnosed with esophageal carcinoma who received radical radiotherapy in our hospital from November 2013 to April 2015 were enrolled as subjects. Target volume was delineated on the CT images and MRI images(T2-weighted and diffusion-weighted fusion images). The MRI-and CT-based IMRT plans were designed using the same dose prescription and dose constraints for organs at risk(OAR). The target volume,prescribed dose,and doses for OAR were compared between the two plans. Results In the two plans, dose distribution and planning parameters met the clinical requirement. The length of lesion,gross tumor volume (GTV),and planning target volume(PTV)defined by 3.0 T MRI were significantly smaller than those defined by CT(P=0.00,0.03,0.03). There were no significant differences in the D 2%,D 98%,D 50%,homogeneity index,or conformity index for primary GTV(PGTV)and PTV-PGTV between the two plans(all P>0.05). Compared with the CT-based plan,the 3.0 T MRI-based plan had a significantly smaller mean dose to the lungs and an insignificantly smaller actual dose to the lungs(P=0.00;P>0.05).There were no significant differences in maximum doses tolerated by the spinal cord or heart between the two plans. Conclusions In terms of target volume delineation and dosimetric parameters, both CT-and 3.0 T MRI-based plans meet the clinical requirement. The 3.0 T MRI-based plan may provide potential benefits for some OAR due to a smaller target volume compared with the CT-based plan.

BACKGROUNDBased on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.

METHODSTwo unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.

RESULTSThe mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.

CONCLUSIONSThe authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Amino Acid Substitution ; Antibodies, Viral ; Antiviral Agents ; pharmacology ; Cells, Cultured ; DNA Mutational Analysis ; Humans ; Interferon Type I ; genetics ; pharmacology ; Interferon-alpha ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; genetics ; pharmacology ; Plasmids ; genetics ; Receptors, Interferon ; metabolism ; Recombinant Proteins

In this experiment, Hca-F or L615 elemene combo-TCV (H-TCV and L-TCV), H-TCV lysates, corynebacterium parvum (CP) were used to immunize 615 and Balb/c inbred mice, and their splenic DCs were prepared and pulsed in vitro with tumor cell lysates (H or L) and TCV lysates (TH or TL). The capacities of DCs to stimulate the proliferation of syngeneic nonadherent spleenic cells were tested with MTT assay. The results showed that the splenic DCs from normal mice in vitro pulsed with TH or TL could induced syngeneic noradherent splenic cells to proliferate (P＜0.01), while the H or L pulsed DCs could not. The splenic DCs from H-TCV or L-TCV or TH immunized mice re-pulsed in vitro with TH or TL exhibited stronger stimulating effects than the DCs from normal mice pulsed in vitro for the firth time pulsed with TH or TL (P＜0.01 or P＜0.05); The capacities of DCs to induce proliferation of syngeneic nonadherent splenic cells could be further enhanced by CP immunization, especially when were pulsed with TH (P＜0.01). Normal inbred 615 mice were transferred with DCs pulsed with lysates of elemene TCV (TDCs) or pulsed with lysates of Hca-F tumor cells (HDCs) on day 7 before challenged with lethal dose of live Hca-F cells, significant adoptive immunoprotective effects were seen, with 61.6% tumor inhibition rate and 25% survival in TDC adoptive transfer group. This study indicated that DCs might play a role in the mechanisms of active immunization and pulsing DCs with lysate of elemene combo TCV and isolating DCs from elemene combo TCV immunized mice were useful methods for DCs vaccine preparation.

Elemene is a new anticancer drug isolated from a Chinese traditional medicine Curcuma aromatica. In previous work, we discovered that tumor cell vaccine (TCV) treated with oleum Curcuma aromatica or elemene could induce significant immunoprophylactic effect against a variety of aminal tumor strains and the method of preparation of elemene combo-TCV(EC-TCV) already got China's inventive patent. In this paper we further studied the active immunotherapeutic effect and the possible cellular/molecular mechanisms of EC-TCV immunization. The results were as follows:(1) EC-TCV immunization showed significant therapeutic effects (P＜0.05) against murine Ca761 syngeneic mammary carcinoma (H-2k) and HCa-F allogeneic hepatic carcinoma (H-2-) models; (2) The spleen cells of Hca-F EC-TCV immunized mice displayed higher cytotoxicity and IL-12 level while the secretion of IL-10 was decreased (P＜0.05); (3) Similar to heat shock, elemene(E), mitomycin C(MMC) and glutaraldehyde (G) could act alone as stressor, and induce significant changes of the expression of membrane heat shock proteins(HSP70 or/and HSP90) on L615 leukemia and HCa-F hepatoma cells and the EC-TCVs (E+MMC+G treated in combination) showed the highest level of membrane HSPs expression (P＜0.05 or P＜0.01 );(4) The HSP70-peptide complex isolated from HCa-F EC-TCV through ADP-agarose affinity chromatographic system could induce active immunoprotection against lethal dose challenge of HCa-F hepatic cancer cell but could not protect against the cross challenge of lethal dose of L615 leukemia. The results indicated that the immunoprotective effect of EC-TCV was in some extent tumor-specific, MHC-nonrestricted, and HSPs might play an important role in its molecular mechanisms.

Objective:Normal murine DCs were pulsed with complex of tumor antigen from elemene-combo tumor cell vaccine-heat shock protein 70 of BCG (H TA-HSP70 BCG).Their proliferation and antigen presenting function were evaluated.Methods:The dendritic cells(DCs)were cultured in complete media containing GM-CSF and IL-4 and pulsed with H TA-HSP70 BCG,H TA or HSP70 BCG.Their proliferation and stimulating effects on spleen nonadherent cells were evaluated with MTT assay.Their capability of endocytosing FITC labeled dextran was assayed with FACscan,morphological changes of DC were observed in electron microscope.Results:Proliferation index of DCs pulsed with H TA-HSP70 BCG was 2.107?0.013,proliferation index of DCs pulsed with H TA-HSP70 BCG mixed with normal nonadherent spleen cell was 1.927?0.073.The percent of DCs endocytosed FITC labeled dextran was 58.61%.Above changes were more significant than those of DCs pulsed with H TA or HPS79 BCG.Conclusion:H TA-HPS70 BCG had more potent activity to pulse DC and strengthen antigen presenting of DC.

Objective: Identification of the genes specially expressed in tumor cell but not in normal cell is important for understanding the molecular mechanisms of carcinogencsis. This study will focus on identification of differentially expressed gene fragments in human stomach cancer. Methods: By using the new developing mRNA differential display (DD) technique, genes fragments differentially expressed in stomach cancer tissues from a patient and the adjacent normal tissues beyond the tumor mass were studied. Results: Two differentially displayed complementary DNA fragments from stomach cancer tissues, scgl and scg2 (stomach cancer-associated gene, scg), cofirmed by Northern Blot, were cloned and sequenced. The nucleotide length of scgl is 194 base pairs and that of scg2 is 343 base pairs. After searching against GenBank databases by BLASTN, neither scgl nor scg2 had significant homological gene sequences with the known genes. Conclusion: These results suggested scgl and scg2 might be complementary DNA fragments of novel genes expressed in stomach cancer tissues, but not in normal tissues and may play a role in the occurrence and development of stomach cancer. Further characterization of full-length of these two complementary DNA fragments will be continued.