AIM:To observe whether modified epitopes from pancreatic tumor antigen mucin 4 (MUC4) have HLA-A2-restricted antitumor ability.METHODS:RT-PCR and Western blot were used to identify the expression of MUC4 in the pancreatic tumor cell lines CAPAN-2 and ASPC-1.HLA-A2 epitopes from MUC4 protein were predicted by the software of NetCTL 1.2, BIMAS, SYFPEITHI and IEDB.The modified peptides from MUC4 containing HLA-A2 were obtained by replacing anchor residues of the binding anchor motifs.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecule was evaluated by T2 binding assay.ELISPOT assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T-lymphocytes (CTLs) and release of IFN-γ.The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The expression of MUC4 was observed in the CAPAN-2 cells and ASPC-1 cells.The candidate peptides P1944-1Y, P1944-2L, P1944-1Y2L, P2004 and P2004-1Y9V showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P1944-1Y2L and P2004-1Y9V had significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay showed P1944, P1944-1Y2L, P2004 and P2004-1Y9V were able to induce specific CTLs and more amounts of IFN-γ were released.ELISPOT assay showed that significantly more amounts of IFN-γ released by P1944-1Y2L and P2004-1Y9V were observed than the native peptides.The CTLs induced by P1944, P1944-1Y2L, P2004 and P2004-1Y9V lyzed the CAPAN-2 cells.P1944-1Y2L and P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell line CAPAN-2 than the native peptide-specific CTLs.CONCLUSION:Compared with the native peptides, modified epitopes P1944-1Y2L and P2004-1Y9V have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the anti-tumor immunity of modified epitopes P1944-1Y2L and P2004-1Y9V is stronger than that of the native peptides.The peptides P1944-1Y2L and P2004-1Y9V are excellent HLA-A2-restricted CTL epitopes from tumor antigen MUC4, which could serve as new candidates towards antitumor peptide vaccines.

Objective To indentify the relationship between hepatitis B virus covalently closed circular DNA (HBV cccDNA) and postoperative recurrence of HBV in HBcAb positive liver donors by detecting HBV cccDNA in liver from HBcAb positive liver donors.Method Eighty-five of 1200 patients underwent liver transplantation for hepatitis B-related end-stage liver disease in our hospital from January 2007 to January 2010 were retrospectively analyzed.According to the situation of HBV infection in donor liver,the recipients were divided into 3 groups:(1) the experimental group (livers positive for HBcAb,and negative for HBsAg,n =40),(2) control group (livers positive for HBsAg,n =15),and (3) normal group (donor livers without HBV infection,n =30).HBV cccDNA of donors was detected by fluorescence quantitative PCR.Serum HBV and HBV DNA were regularly tested,and biopsy was done in those positive for HBsAg or HBV DNA to confirm HBV recurrence.The relationship between HBV cccDNA and postoperative recurrence of HBV in HBcAb positive liver donors was analyzed.Result The positive rate of cccDNA was 30％ in experimental group (12、40),73.3％ in control group (11/50),and 0 (normal group).The rate of HBV recurrence in experimental,control and normal groups was 10％ (4/40),80％ (12/15) and 3.3％ (1/30) respectively.The rate of HBV recurrence in the experimental group of cccDNA (+) and cccDNA (-) was 7.5％ (3/40),and 2.5％ (1/40).Conclusion The cccDNA in HBcAb positive donors may be one of the risk factors in HBV recurrence of hepatitis B-related liver transplantation patients.The screening of HBV cccDNA in the donor livers positive for HBcAb before liver transplantation is recommended to reduce the positive HBV recurrence and expand the pool of liver donors for patients with HBV-related liver disease.

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.

Healthcare Big Data and its driven real-world study (RWS) have increasingly become important sources of evidence for medical decision-making.Drawing on successful international experiences, China′s neonatal-perinatal field has established national or regional neonatal collaboration networks to conduct various forms of clinical scientific researches, continuously improving the treatment and care level for preterm infants.How to construct a scientific clinical research methodology system based on clinical issues in China′s neonatal field and reliable real-world data is currently a pressing issue that needs to be addressed.This article discussed the methodological exploration for advancing neonatal clinical scientific research through regional collaboration networks.

Objective: To examine the Quality of life among school-aged children with dyslexia in target city and to provide scientific evidence for improving the quality of life of children with dyslexia. Methods: By using cluster sampling,students from grade 3 to grade 6 from 6 primary schools in a middle-sized were selected and administered with questionnaire survey. According to the criteria of dyslexia, dyslexic children and non-dyslexic children were identified and the difference of the Quality of Life was compared. Results: Totally 3 673 children were collected, and 119 of them were identified as dyslexia(3.24%).The prevalence of dyslexia differed by gender,grades,educational level of parents(χ2=24.77,11.75,18.50,9.79,P<0.05). The Quality of Life which below the average proportion accounted for 30.3% of dyslexic children and 16.7% of normal children. Quality of life scored signiticantly different between dyslexic children and non-dyslexia children, including psychosocial functioning domain(134.54±30.88)(143.49±32.53), physical and mental health domain(2.71±0.84)(2.92±0.81) vs (2.83±0.90)(3.06±0.87), the living satisfaction domain(2.95±0.87)(3.14±0.87)(t=-6.09，-5.48，-5.44，-4.50，P<0.01),with dyslexic group significantly lower than that of non-dyslexic group. Conclusion The Quality of Life of Dyslexic children was in a poor condition.

Objective To investigate and compare predictive diagnostic value of neutrophil-to-lymphocyte ratio,platelet-to-lymphocyte ratio and mean platelet volume,for early-stage diabetic nephropathy (EDN).Methods 245 patients with T2DM were divided into patients with EDN(EDN group,n=120) and patients without DN (T2DM group,n=125).116 healthy people were also enrolled in our study.Their clinical and biochemical data were collected.Results NLR (2.14±0.73 vs 1.78±0.58 vs 1.46±0.48,P＜0.05),PLR (116.11±36.43 vs 100.65±27.83 vs 90.24±26.17,P＜0.05) and MPV (11.29± 1.16 vs 10.37 ± 1.01 vs 9.78 ± 1.28,P＜0.05) were significantly higher in EDN group and T2DM group than those of the control group.NLR,PLR and MPV were significantly higher in EDN group than those of T2DM group.Logistic regression analysis showed that after correction of diabetes duration,blood pressure,lipid,and so on,NLR(OR=3.77,95％ CI 2.12-6.08,P＜0.01),PLR(OR=1.81,95％ CI 1.02-2.89,P=0.04) and MPV (OR=2.08,95％ CI 1.22-5.04,P=0.01) were all positively correlated with EDN.According to the analysis of ROC curves,the value of NLR for predicting EDN was higher than PLR and MPV.The cut off value of NLR for predicting EDN was set at 1.82,with the sensitivity of 79.20％ and the specificity of 68.60％.Conclusion NLR has the greatest diagnostic value for EDN,patients whose NLR is over 1.82 need further test in order to diagnose and therapy DN earlier.

Objective:To investigate the clinical diagnosis, treatment, and genetic variations of neonates with congenital hyperinsulinism (CHI).Methods:The clinical data of CHI newborns admitted to the Provincial Hospital Affiliated to Shandong First Medical University from September 2018 to April 2022 were retrospectively analyzed.Results:Four cases of CHI were included, three of whom were full-term infants and all were macrosomic, while one was a premature infant. One infant was born to a mother with gestational diabetes mellitus, and 1 had a family history of hypoglycemia. All the 4 patients presented with weak response, 3 with drowsiness, 1 with hypotonia and 1 with convulsions. Cranial MRI indicated abnormal signals in the occipital lobe cortex in 1 case. Gene sequencing revealed homozygous variation c.799C>G in KCNJ11 gene for 1 case, and heterozygous variations c.4477C>T, c.3540C>G, c.683G>A and c.4536C>A in ABCC8 gene for 3 cases respectively and all these variations were identified as pathogenic mutations. Notably, the c.799C>G variant in KCNJ11 gene as well as the c.3540C>G and c.4536C>A variants in ABCC8 gene were reported for the first time. Among infants with ABCC8 gene variations, two showed no response to diazoxide treatment while one patient with KCNJ11 gene variation responded effectively. The parents of the patient with hypoglycemic brain injury gave up treatment. Three other cases were discharged from hospital after improvement and followed up to 1 year old. 2 patients had stable blood glucose after ceasing medication, and 1 patient still required intermittent oral glucose to maintain normal blood glucose level.Conclusions:CHI can lead to hypoglycemic brain injury. Clinically, infants large for gestational age or with a family history of diabetes and hypoglycemia should be monitored for blood glucose early after birth, to identify CHI as early as possible and actively treat it. Different gene variants have different therapeutic responses.

Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.