Design and antitumor activity of programmed cell death ligand 1 epitope peptide vaccine / 中国药科大学学报

@#Several programmed cell death protein 1 (PD-1) or its ligand (PD-L1) immune checkpoint blocking antibodies are available for clinical treatment, but only some patients show clinical response, so an alternative strategy for tumor immunotherapy is needed.A therapeutic tumor vaccine targeting PD-L1 is a meaningful attempt.In this study, we designed an epitope peptide vaccine targeting PD-L1, and then screened the immunogenic PD-L1 epitope peptide based on the humanized immune system (HIS) mouse model and further investigated its anti-tumor activity.The results show that the designed and screened PD-L1-B1 epitope peptide vaccine not only successfully induced PD-L1-specific humoral and cellular immunity, but also exhibit anti-tumor activity.In addition, immunotherapy increased T-lymphocyte infiltration of tumors and reshaped the tumor immunosuppressive microenvironment.In conclusion, PD-L1-B1 epitope peptide vaccine exhibits potent anti-tumor activity and may be an effective alternative immunotherapeutic strategy for patients insensitive to PD-1/PD-L1 blockade.

Design and oral hypoglycemic activity of novel oral hypoglycemic peptide ODA / 中国药科大学学报

@#Incretin promotes insulin secretion through a glucose-dependent mechanism, involving glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). Therefore, their correspondingly specific receptors GLP-1R and GIPR are suitable targets for the treatment of type 2 diabetes. Based on the oral hypoglycemic peptide OHP2 designed by our team, we further designed a new oral hypoglycemic peptide, ODA to reduce glucose. Compared with OHP2, ODA exhibited better lipophilicity as well as the enhanced endocytosis and transcytosis in Caco-2 cells. In addition, ODA remained the ability to activate GLP-1R and enhanced the binding ability to GIPR. The hypoglycemic efficacy of the low-dose ODA (0.53 mg/kg) is comparable to that of OHP2 (1.06 mg/kg). These results indicated that ODA could be a new oral drug with potential for the treatment of type 2 diabetes.

Exploration of reform paths of high-level talents training in clinical pharmacy / 中国药房

OBJECTIVE To help to realize high-quality development of clinical pharmacy education in China by exploring new reform paths of high-level talents training in clinical pharmacy. METHODS The concept definition and key links of high-level talents training in clinical pharmacy were consulted by expert consultation， and the literature analysis and empirical methods were used to prepare a questionnaire to conduct online research on clinical pharmacy professionals in universities and hospitals. RESULTS A total of 637 effective electronic questionnaires were received. Totally 95.13% of the respondents believed that the cultivation of high-level talents in clinical pharmacy was very or relatively important； 51.96% expressed different degrees of dissatisfaction with the current situation of training； 88.85% regarded “rational clinical use of drugs” and 88.70% regarded “clinical research of drugs” as one of the main training objectives and service orientation； precision pharmacy， evidence-based pharmacy， medication therapy management， therapeutic drug monitoring and evaluation， clinical discovery and evaluation of new drugs were considered as important core specialty knowledge and competences， which were different from those high-level talents in clinical medicine；62.01% agreed that “academic degree+professional degree” dual degree training was the main degree type for high-level talents training in clinical pharmacy； 79.28% thought it was very necessary or relatively necessary to adopt the “long schooling” education mode； 98.58% thought that the core courses of clinical pharmacy such as clinical pharmacotherapy were very important or relatively important；59.81% believed that college or department of clinical pharmacy was the most important educational management organization that played the most important role； 87.13% agreed with the dual tutor structure of “college teachers+ pharmacists” and “pharmacists+physicians”. CONCLUSIONS Starting with the target orientation， core knowledge and competence， academic degree system， curriculum system and faculties， it is necessary to speed up the exploration of new reform paths of talent training and build a high-level talent training system of clinical pharmacy with Chinese characteristics and world level.

Effects and mechanisms of microRNA-23b on neuronal apoptosis induced by Aβ25-35 / 中国药科大学学报

@#In this study, the effects and mechanisms of miR-23b in the AD cell model were explored. Aβ25-35 was used to induce neuronal injury model, and cell viabilities were detected by MTT assay. The effect of miR-23b on the apoptotic levels of Aβ25-35-induced SH-SY5Y cells was analyzed using Annexin V-FITC/PI detection kit. The effect of miR-23b on the mitochondrial membrane potential of Aβ25-35-induced SH-SY5Y cells was examined using JC-1 fluorescent probe. The levels of cell apoptosis-related proteins and autophagy-related proteins were detected by Western blot. The results showed that miR-23b could alleviate the apoptosis and the abnormal mitochondrial membrane potential in SH-SY5Y cells induced by Aβ25-35.This study suggested that miR-23b may attenuate Aβ25-35-induced neuronal apoptosis by regulating apoptosis and autophagy-related pathway.

Design and antitumor activity of immune checkpoint B7-H3 epitope vaccine / 中国药科大学学报

@#B7-H3 is an immune checkpoint molecule overexpressed on the surface of a variety of tumors, and is is an ideal target for tumor immunotherapy. In this study, nitrolated T cell epitope designed in the early stage of the laboratory was used to construct an epitope vaccine that can target immune checkpoint B7-H3. The vaccine can significantly inhibit tumor growth in the CT26 colon cancer model, and has a significant synergistic effect with the PD-L1 protein vaccine. B7-H3 vaccine can increase the proportion of CD4+ T cells in splenic T lymphocytes and the proportion of CD8+ T cells in tumor-infiltrating T lymphocytes, while reducing the proportion of suppressor Treg cells in tumor-infiltrating CD4+ T lymphocytes, which effectively improves tumor immunosuppressive microenvironment. Research results suggest that the B7-H3 epitope vaccine can be used as an effective tumor vaccine candidate molecule.

Advances of DNA-based nanomaterials in tumor therapy / 中国药科大学学报

@#Antitumor drugs usually have deficiencies such as poor water solubility, low targeting, poor stability, and difficulty in being taken up by tumor cells. The development of an ideal drug delivery vehicle is still an urgent problem to be solved in the field of cancer therapy. Due to their excellent sequence programmability, biocompatibility and biodegradability, DNA-based nanomaterials have been widely used as drug delivery vehicles for cancer treatment. Numerous studies have shown that DNA nanomaterials can effectively load cancer therapeutic agents, and achieve tumor targeted delivery, efficient cellular internalization as well as stimuli-responsive drug release. Starting from the history and development of DNA nanotechnology, this review illustrates the application progress of DNA nanomaterial as drug delivery vehicle in chemotherapy, gene therapy, immunotherapy and photodynamic therapy, and the future development is prospected so as to provide some reference for other researchers in this field.

Effects of different immunogenic amino acids in PD-L1 vaccine on the differentiation of T cell subsets / 中国药科大学学报

@#To compare the effects of endogenous 3-nitrotyrosine and non-natural 4-nitrophenylalanine in PD-L1 vaccine on the differentiation of T cell subsets, two immunogenic amino acids were introduced into the same site of PD-L1 vaccine. Two PD-L1 mutants with 3-nitrotyrosine and 4-nitrophenylalanine were obtained, respectively, using genetic code expansion technology. Mice were immunized with these two mutants, and their effects on the differentiation of T cell subsets in spleen were analyzed. The results of flow cytometry showed that the introduction of 4-nitrophenylalanine in PD-L1 vaccine could promote the polarization of Th1 cells while reducing the proportion of Treg cells; the introduction of 3-nitrotyrosine had no effect on the polarization of Th1 cells, while significantly increasing the proportion of Treg and Th17 cells. The introduction of both into PD-L1 vaccine could promote the response of CD8+ T cells in spleen, and the response of PD-L1 mutant containing 4-nitrophenylalanine was stronger. In summary, the non-natural 4-nitrophenylalanine is more suitable for the design of tumor vaccines as compared with endogenous 3-nitrotyrosine.

Study on the Medical Insurance Budget Impact Analysis Guideline in Some European Countries and Its Enligh- tenment to China / 中国药房

OBJECTIVE：To study the m edical insurance budget impact analysis （BIA）guidelines or nomative documents of some European countries ，and to provide the suggestions for the formulation and implementation of medical insurance BIA guidelines in China. METHODS ：Medical insurance BIA guidelines or related documents in European countries such as Ireland ， France，Poland，Belgium and UK were retrieved to summarize and comparatively analyze the general analysis framework and special specification. The formulation of medical insurance BIA guideline in China and the suggestions were put forward. RESULTS & CONCLUSIONS ：The above-mentioned medical insurance BIA guidelines or documents of the five European countries generally study the impact of the cost of health technology on resources within 3-5 years from the perspective of budget holders. The analysis framework of the guidelines or documents is basically the same ，but the guidelines or documents are adjusted according to the characteristics of national health system in terms of the positioning of medical insurance BIA ，the scope of cost data inclusion ， model design ，population subgroup analysis and so on. For example ，Ireland had special requirements on cost data inclusion ， sensitivity analysis and data source ，while France had detailed regulations on medical insurance BIA model ，sensitivity analysis and presentation of medical insurance BIA results. Our country should pay attention to the role of medical insurance BIA in medical and health decision-making ，formulate China ’s medical insurance BIA guidelines to standardize empirical research ，and combine the characteristics of China ’s health system when formulate the guideline. It is suggested that China ’s medical insurance BIA guidelines should at least include research perspective ，research time limit and discount ，reference situation ，target population ， cost，market share ，data source ，uncertainty analysis and other overall framework or basic elements to ensure the smooth operation of medical and health funds.

A novel human immune system mice model for assessing the immunogenicity of cancer vaccines / 中国药科大学学报

@#Human immune system(HIS)mice are usually used to evaluate the ability of tumor vaccines to induce cytotoxic T lymphocyte(CTL)effects, but they failed to accurately reflect the ability of cancer vaccines to induce humoral immune responses. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and co-transplanted into NCG mice with in vitro differentiated dendritic cells(DCs)to establish a DC-HIS mouse model. In DC-HIS mice, co-transplanted antigen-presenting cells(HLA-DR+CD11c+)could colonize the spleen of model mice. Moreover, co-transplantation of DCs significantly increased the proportion of activated human CD4+ T/CD8+ T cells and B cells in HIS mice, indicating that DC-HIS mice could better mimic the human immune responses. The immunogenicity of the targeted HER2 protein vaccine(NitraTh-HER2)was evaluated using the DCs-HIS mice. The results showed that the NitraTh-HER2 vaccine was able to induce the production of HER2-specific human IgG antibodies with a significant antibody-dependent cell-mediated cytotoxicity(ADCC)effect and the lysis rate of target cell SK-BR-3 reached 47. 1%. The NitraTh-HER2 vaccine was able to produce antigen-specific CTL effect, and the lysis rate of target cell SK-BR-3 reached 14. 6%. Taken together, the DC-HIS mouse model provides an effective method for predicting the immunogenicity of human tumor vaccines.

Design and antitumor effect of nitrated T helper cell epitope vaccine CD47-NitraTh / 中国药科大学学报

@#Using the genetic code extension technology, the immunogenic amino acid, p-nitrophenylalanine, was introduced into the universal T cell epitope and then fused with the fragment of the extracellular region of the immune checkpoint molecular CD47(19-140)to construct a vaccine targeting CD47. The CD47-NitraTh vaccine elicited high titer antibody in BALB/c mice, significantly inhibited CT26 colon cancer cells growth, and increased the ratio of spleen CD4+ T cells and CD8+ T cells. Meanwhile, it promoted the polarization of naï ve T cells to Th1 cells. Notably, CD47-NitraTh not only increased the proportion of tumour-infiltrating lymphocytes but also reduced the proportion of Treg cells in tumour tissues, which means that CD47-NitraTh vaccine can remodel the tumour immunosuppressive microenvironment. The results of this study suggested that CD47-NitraTh can be used as an effective tumour vaccine candidate.

Establishment of a high-throughput analysis method for the identification of key proteases of peptide drugs in vitro / 中国药科大学学报

@#In order to establish an effective analytical method to detect the key proteases which affect the metabolism and the plasma half-life of peptides in vivo, a method to analyze the key proteases of peptide drugs based on high performance liquid chromatography in vitro was established. The general enzymatic reaction conditions in vitro were as follows: pH was 7. 8 or 9. 0 and the buffer system was 0. 01 mol/L PBS or 50 mmol/L Tris buffer. The results of pramlintide detected by this method showed that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 had the strongest hydrolysis on pramlintide. The result was consistent with that determined by microscale thermophoresis, which indicated that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 were the key proteases of pramlintide. This analytical method provides the basis for high-throughput stability screening of peptides and can be used to analyze key proteases of peptide drugs. It can also provide guidance for optimizing the protease stability of peptide drugs.

Research advances of bispecific antibody drugs in tumor therapy / 中国药科大学学报

@#Tumor immunotherapy is currently the new direction for the treatment of cancer. Bispecific antibody can bind two different antigens, so the development prospect in the field of tumor treatment is very attractive. The most compelling trifunctional antibody and bispecific T-cell engager in bispecific antibodies have been marketed separately, with representative drugs as catumaxomab and blinatumomab, respectively. So far, nearly 100 antitumor bispecific antibody drugs are undergoing clinical trials and in-depth understanding of their mechanisms of action will provide more powerful solutions for cancer treatment. This review summarizes the progress of catumaxomab, blinatumomab and current highly promising bispecific antibody drugs, for the further development and application of tumor therapy.

Quality Evaluation of the Literatures about Medical Insurance Budget Impact Analysis in China and the United States / 中国药房

OBJECTIVE： To provide experience and reference for the study of medical insurance budget impact analysis （BIA） in China. METHODS： Retrieved from PubMed， ProQuest， CNKI， Wanfang database and CBM， related literatures about medical insurance BIA research in China and the United States were collected since the establishment of the database. The basic information， analysis results and data sources were summarized and sorted out， and descriptive analysis of the included literature was carried out on basis of seven key elements such as model design， research perspective， treatment cost， reference scenario， target population， research time limit and discount/inflation， sensitivity analysis. RESULTS： A total of 72 literatures were included in this study， involving 24 （33.33％） studies in China， 48 （66.67％） studies in the United States； the indications of 45 studies were chronic diseases （62.50％）， and those of 21 studies were acute diseases （37.50％）. Among the research methods， 49 studies （68.06％） used BIA alone and 23 studies （31.94％） adopted BIA combined with pharmaceutical economics. In terms of model design， 50 studies （69.44％） adopted cost calculation models. In terms of research perspective， 60 studies （81.94％） were based on the perspective of medical insurance department research. In the calculation of treatment cost， 69 studies （95.84％） included drug cost. In terms of reference scenarios， 61 studies （84.72％） compared the economics of different drug-based treatment groups. For target population， only 31  （43.06％） studies used real world data. In terms of research duration and discount/inflation， 14 studies （19.44％） used treatment or length of hospitalization to indicate research duration， and 19 studies （26.39％） used discount rate or inflation rate to adjust costs. As for sensitivity analysis， 62 studies （86.11％） conducted sensitivity analysis， of which 49 （68.06％） used single factor sensitivity analysis. CONCLUSIONS： There are still some limitations in medical insurance BIA research literature in China and the United States， such as unreasonable use of data， incomplete coverage of the cost， and unreasonable setting of sensitivity analysis variables. It is recommended that BIA research should standardize data sources to improve the quality of budget evidence quality， reasonably evaluate market size to improve the authenticity of prediction， scientifically set variables and their scope of change to improve the stability of results， establish BIA research paradigms or evaluating standards so as to guide BIA research scientifically.

lmmunogenicity of HER2 vaccine containing p-nitrophenylalanine / 中国药科大学学报

In order to delete the immune tolerance of self-protein HER2 and produce an efficient immune response,the genetic code expansion technique was employed to introduce the immunogenic amino acid p-nitro-phenylalanine into the position 5 ,26 and 79 of the HER2 fragment and HER2 mutants containing p-nitropheny-lalanine were obtained.Prototype and mutant HER2 molecules with a purity of more than 95% were obtained through a nickel affinity column.Immunogenicity analysis of the HER2 vaccine showed that pNO2Phe79HER2 was able to produce high titer cross-reactive antibodies in C57BL/6 mice and that the mouse antisera could recognize SKBR-3 cells with intact HER2 receptor extracellular domain and cleave HER2 highly expressed HER2 +B16F10 cells through ADCC effect.These findings suggested that pNO2Phe79HER2 which incorporates p-nitro-phenylalanine could be used as a tumor vaccine candidate targeting HER2.

Effects of immunogenic HER2 on the differentiation of T-cell in mouse / 中国药科大学学报

In order to investigate the effects of p-nitrophenylalanine on the differentiation of T lymphocyte,human epidermal growth factor receptor 2(HER2)mutants were constructed by incorporating p-nitrophenylalanine to the extracellular domain of HER2.Mice were immunized with HER2 mutant to analyze the effects of p-nitrophenylala-nine on the differentiation of T cell subsets.The results showed that the immunogenicity of HER2 protein was significantly enhanced by incorporation of immunogenic amino acids.HER2 mutants induced production of more anti-WT-HER2 antibody and greater titer of antibody than PBS and wild-type HER2 in the fifth week.The differ-entiation frequency of Th1 and Th2 cells showed no significant difference,but the differentiation frequency of Tfh cell was found to increase from 4％ to 8％,while the differentiation frequency of Treg cell was found to decrease from 5％ to 1％ through flowcytometry analysis.The gene expression of Bcl-6 and FOXP3 were determined by quantitative real-time PCR.The results showed that the expression of Bcl-6 mRNA increased about 2 fold,while the expression of FOXP3 mRNA could be decreased to about 50％ in HER2 mutant-immunized group.The secre-tion level of IL-21 in serum was also detected to increase from 4 to 28 pg/mL in HER2 mutant-immunized group.The above results confirm that HER2 mutants containing p-nitrophenylalanine can cause an efficient immune response by activating Tfh cell differentiation.

Advances of combined immunotherapy in tumor / 中国药科大学学报

@#Immune checkpoints are inhibitory signaling pathways in the immune system that maintain balance with co-stimulatory molecules, maintain tolerance to their own tissues, and avoid autoimmune responses. The development of tumors is accompanied by the upregulation of co-suppressor molecules and related ligands, causing the decline or exhaustion of T cell functions, which makes tumor cells excape immune surveillance. The development of monoclonal antibodies against inhibitory receptors and ligands to regulate T cell activity and improve antitumor immune responses have achieved promising clininal results. Combined treatment of immunomodulatory signaling pathways also showed a certain of synergy. This article summarizes the evaluation of combined tumor immunotherapy strategies and synergies, and outlooks the feasibility of combined immunotherapy and the selection of immunization strategies.

Modification of mPEGylated novel bifunctional fusion protein GAD and its propenty / 中国药科大学学报

In order to solve the difficucties of renaturation and immunogenicity of new bifunctional fusion protein GAD,inclusion bodies of GAD were modified by PEG-maleimide.Conformational changes of the modified GAD were compared by circular dichroism and tryptophan fluorescence spectroscopy.The biological activity was verified by oral glucose tolerance test and lipid scavenging.The results showed that PEG-maleimide completed the specific-point modification of GAD,and improved its refolding efficiency.The secondary and tertiary structures of mPEGylated GAD were consistent with that of GAD.PEG-GAD has significant hypoglycemic and lipid-lowering effects (P ＜0.001) with longer half life in vivo and lower immunogenicity (P ＜0.01).This study provides effective strategies for the development of strongly hydrophobic peptide drugs.

Advances of lncRNA in immune cells and autoimmune diseases / 中国药科大学学报

Long non-coding RNA (lncRNA) is a newly identified non-coding RNA subfamily with various regulatory functions.Recent evidence has shown the fundamental role of long noncoding RNAs in affecting the development of diseases at different levels,from gene modification,transcriptional regulation to protein transla tion.The study on lncRNA has made great progress in the studies of genomic imprinting,cancer diseases and neurodegenerative disorders,while the research relevant to autoimmune diseases has just staaed recently,However,many lncRNAs have been identified to involve in immune cells proliferation,differentiation and maturation,acting as the key regulators in immune homeostasis and autoimmune diseases.This review is focused on the advances of lncRNA in immune cells and autoimmune diseases.

Protective effects of p-nitrophenylalanine-containing BAFF vaccine on lupus nephritis model mice / 中国药科大学学报

In order to verify whether p-nitrophenylalanine-containing BAFF vaccine can be used as a candidate molecule for the treatment of autoimmune diseases with BAFF over-expression,a soluble mutant of B cell activating factor belonging to the TNF Family (smBAFF) and its pNO2Phe mutant(pNO2Phe65smBAFF),which site specific incorporated pNO2Phe at position 65 of smBAFF,were expressed and purified.In order to evaluate the feasibility of using pNO2Phe65 smBAFF to treat BAFF-over-expressed autoimmune diseases,we investigate its Lymphocyte-stimulating capacity,immunogenicity and inhibitory effect of serum on biological activity of natural BAFF.The pharmacological activity of pNO2Phe65 smBAFF was evaluated using a cGVHD(graft-versus-host disease) induced SLE mouse model.Results indicated that pNO2Phe65 smBAFF,could bind to mouse lymphocytes but could not promote the proliferation of mouse lymphocytes.Moreover,the incorporation of pNO2Phe significantly increased the immunogenicity and induced cross-antibody,which can inhibit the biological activity of natural BAFF.In cGVHD induced SLE mouse model,pNO2Phe65 smBAFF can significantly reduce the symptoms of the disease and play a therapeutic role.Therefore,pNO2Phe65 smBAFF can be used as a candidate molecule for the treatment of autoimmune diseases with BAFF over-expression.

Molecular dynamics simulation and analysis of BAFF which is incorporated with p-nitro-L-phenylalanine / 中国药科大学学报

In order to study the structure-function relationship in the protein which is incorporated with p-nitro-L-phenylalanine,the method of MD(Molecular Dynamics) simulation was established and successfully used in the analysis of protein which contains p-nitro-L-phenylalanine.The force field of CHARMM can only stimulate protein with natural amino acid in NAMD.Compared with phenylalanine,p-nitro-L-phenylalanine just has one more group of nitro.If the parameter of group of nitro was defined,the protein containing p-nitro-L-phenylalanine can be simulated.CGenFF-paramchem was used to calculate the energy and topological structure of p-nitro-L-phenylalanine' s new bonds (r),angles (θ),dihendrals (φ) and improper angle (ψ).And then the new defined parameter and topology information was input into the related parameter files and topology files in CHARMM.On the basis of correct parameter,NAMD can successfully simulate the modified BAFF(B lymphocyte stimulator) which contains p-nitro-L-phenylalanine.The changes in structure indicated that there might be new B cell epitopes.The temperature distribution of each frame in the process of dynamics stimulation was in accord with normal distribution,which proved the defined force field parameters was feasible.The RMSD of whole protein solution systemis 2.5.Calculate each resides' RMSF in BAFF,the RMSF of p-nitro-L-phenylalanine's residue is 3.7,which is obviously higher than that of the other residues in β-pleated sheet,and close to the loop rings,indicate that there might be variation in the area of p-nitro-L-phenylalanine residue and might produce new comformational epitopes.The results of MD stimulation will guide the immunogenicity experiments of p-nitro-L-phenylalanine modified proteins.