To identify novel HLA-A2-restricted CTL epitopes derived from the tumor antigen MAGE-A3.Methods:The CTL epitope candidates were predicted by using the motif prediction method combined with the secondary anchor residues plot. It was established that the molecule model of CIL epitope candidates bound to the HLA-A2 molecule and of the HLA-A2-peptide complex by molecule modeling. Four selected candidates were assayed by the standard 51 Cr release assay to determine their abilities of inducing the generation of specific CTLs. Results: Among them, the pepnde MAGE-A3201-209 could effectively induce specific Oils and the CITs could lyse not only the melanoma cell line LB373-MEL but also the murine mastocytoma cell line genetically modified with the human HLA-A2 gene and pulsed with MAGE-A32oi.2cs ? Conclusion; MAGE-AS201-209 is a novel CTL epitope presented by HLA-A2. This study provides experimental basis for design and study of tumor therapeutic peptide vaccines based on the tumor antigen MAGE-A3.

Objective To explore how to trigger an HLA Ⅰ restricted CD8 + T cell response to exogenously synthesized peptides in vitro . Methods A new panel of therapeutic peptides based on the immunodominant B and CTL epitopes of HBV PreS 2 region and HBcAg and the tetanus toxoid common T helper epitopes were synthesized by Merrifield solid phase peptide synthesis, and HLA A2 + human PBMCs were used to investigate the immunological properties of the mimetic peptides. Results The results demonstrated that the peptides could trigger vigorous CD8 + HBV specific CTL responses in vitro specifically and effectively. Conclusion The results reveal that T helper plus B epitopes designing with the introduction of short and flexible linker can remarkably improve the immunogenicity of short peptides and hence produce effective CTL responses in vitro .

Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.

Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.

Objective:By reviewing and analyzing the patent of Traditional Chinese Medicine compound for the prevention and treatment of chronic complications of diabetes, this paper aims to analize the patent of Traditional Chinese Medicine compound and medication rules for the prevention and treatment of diabetes complications with data mining technology.Methods:Based on data mining technology, this paper searched for the patent of Traditional Chinese Medicine compound that could prevent and treat chronic complications of diabetes with SOOIP (Intellectual Property Big Data Center) website, and analyzed the application trends, number , categories, etc. Then IBM SPSS Modeler 18.0 software was used for correlation analysis, and finally the medication and compatibility of the Chinese medicine prescriptions are summarized.Results:There were all together 307 patents, and the number of patent applications for the prevention and treatment of chronic complications of diabetes with Chinese medicines has increased before 2015. Most patents in classification belongs to A61P. China accounts for the majority of the global total applications, of which Shandong province accounts the most. The applicants are mostly individuals and enterprises. The categories commonly used in patent applications are mainly oral drug combinations; The Astragali Radix, Puerariae lobatae Radix, Rehmanniae Radix, Coptidis Rhizoma, Salviae miltiorrhizae Radix et Rhizoma are the most commonly used application. The Traditional Chinese Medicine patent mostly has sweet taste, warm in property, and channel-tropism of medicine is mostly liver, as well as the liver is most associated with bitterness in taste. The commonly used couplet medicines are Puerariae lobatae Radix-Astragali Radix, Puerariae lobatae Radix-Rehmanniae Radix, Astragali Radix-Coptidis Rhizoma. Conclusion:The number of such patents applied for in China is small, and the regional development is unbalanced; Data mining technology can be used to discover the compatibility rule of Chinese patent prescription prescription for diabetes prevention and treatment, so as to provide reference for clinical optimization of prescription, improvement of curative effect and development of new drugs for treatment of diabetic complications.

【Objective】 To evaluate the association between prophylactic plasma transfusion and postoperative bleeding rate in critically ill patients undergoing different invasive procedures. 【Methods】 The information of ICU patients who received different invasive procedures from January 2019 to December 2019 in 6 tertiary hospitals in China were retrospectively investigated. The inclusion criteria of patients were as follows: age ≥ 18 years; received invasive procedures; INR ≥ 1.5 within 72 hours before surgery. Exclusion criteria were patients with incomplete case records. The patients finally included in the study were divided into prophylactic plasma transfusion group and non-prophylactic plasma transfusion group according to their plasma transfusion status. The outcome variable was the incidence of invasive procedure-related bleeding within 48 hours after different invasive procedures. 【Results】 A total of 407 patients underwent invasive procedures, and 362 patients were finally included in this study after excluding 45 patients with incomplete case records. The proportions of prophylactic plasma transfusion in different types of invasive procedures were central venous catheterization (46/146, 31.5%), thoracentesis (13/37, 35.1%), bronchoscopy (8/31, 25.8%), tracheal intubation (9/38, 23.7%), arterial catheterization (9/50, 18.0%) and others (13/60, 21.7%). The bleeding rates showed that different invasive procedures presented no statistical difference between the groups received plasma transfusion or not. In the prophylactic plasma transfusion group, the bleeding rate of arterial catheterization (4/9, 44.4%) was the highest, but all were potential bleeding, followed by tracheal intubation (4/10, 40.0%) and central venous intubation (16/46, 34.8%), with a higher rate of significant bleeding. 【Conclusion】 Prophylactic infusion of plasma did not reduce the bleeding rate after different invasive procedures, but prospective studies are needed to further confirm the conclusion; this study also provides a certain data basis for later prospective studies.