This study aims to screen the diagnostic biomarkers for fecal antigen of Echinococcus granulosus(E.granulosus)in dogs with high specificity and sensitivity.The sheep-derived EgPSC artificially infected dogs were collected,and the negative and positive fecal samples of dogs with E.granulosus were prepared by arecoline hydrobromide leakage method.Polyclonal antibody,negative fecal antigen-polyclonal antibody conjugates and positive fecal antigen-polyclonal antibody conju-gates were purified by ammonium sulfate precipitation and affinity chromatography,three groups of samples were detected by ELISA and Western blot,LC-MS/MS and bioinformatics analysis were performed on the three groups of samples.The positive fecal antigen-polyclonal antibody con-jugate was used as the treatment group,the polyclonal antibody and the negative fecal antigen-polyclonal antibody conjugates were used as the control groups to screen the unique peptides of the treatment group.ELISA and Western blot showed that only the positive fecal antigen-polyclonal antibody conjugates were positive.According to LC-MS/MS and bioinformatics analysis,11 unique peptides were screened out only in the treatment group.Among them,3 proteins were related to E.granulosus,namely dysferlin,integrator complex 9 and diagnostic antigen gp50,which were mem-brane-associated proteins,INT complex components and diagnostic antigens.This study has pre-liminarily screened out three candidate canine E.granulosus fecal antigen diagnostic markers,pro-viding a reference for further exploration of diagnostic standards for E.granulosus,screening of echinococcosis target genes,and vaccine development.

Objective:To develop and validate a novel PET tracer, N-cyclohexyl-4-((2, 4-dichlorophenyl)(4-(fluoro- 18F)phenyl)methyl)piperazine-1-carboxamide ( 18F-SQKJ-2), targeting cannabinoid type 1 (CB1) receptors for diagnosing psychiatric disorders associated with fear memory. Methods:18F-SQKJ-2 was prepared using a nucleophilic substitution radiochemical synthesis method. For the CB1 receptor blocking experiment, 7 ICR mice were divided into blocking group ( n=4; rimonabant for blocking treatment) and control group 1 ( n=3; no rimonabant blocking treatment). The affinity and specificity of 18F-SQKJ-2 for CB1 receptors were analyzed based on the differences in 18F-SQKJ-2 uptake (percentage injected dose per gram of tissue, %ID/g) by various organs between two groups. The metabolic stability of 18F-SQKJ-2 in vitro was studied using animal tissue homogenates. Ten C57 mice were used to establish fear memory mouse models (fear group, n=6; control group 2, n=4), and the percentage of freezing time was compared between 2 groups. MicroPET scans were used to detect the intracranial distribution of 18F-SQKJ-2, and the relative uptake in each brain region compared to total brain uptake was calculated. Statistical analysis was conducted to compare the differences in CB1 receptor relative total brain uptake in fear-related brain regions between 2 groups. Independent-sample t test and Mann-Whitney U test were used to analyze the data. Results:18F-SQKJ-2 was successfully synthesized with a radiochemical purity ≥98.0% and a corrected radioactive yield of (12.3±6.0)%( n=4). In vitro metabolic stability experiments showed that 18F-SQKJ-2 was basically stable in the liver, blood, and brain within 60min. The CB1 receptor blocking experiment demonstrated that the uptake of 18F-SQKJ-2 in the brains of mice in blocking group was significantly lower than that in control group 1 ((0.95±0.28) vs (3.44±1.16) %ID/g; t=-3.57, P=0.023). The percentage of freezing time in fear group was significantly higher than that in control group 2 (43.28%(39.46%, 52.93%) vs 2.74%(1.52%, 4.85%); Z=-2.45, P=0.010). 18F-SQKJ-2 microPET imaging showed that the uptake of 18F-SQKJ-2 in the cerebral cortex of mice in fear group was significantly increased compared with that in control group 2 ((5.83±0.47)% vs (5.00±0.52)%; t=2.42, P=0.046). Conclusion:18F-SQKJ-2 is successfully prepared with acceptable radiochemical purity and metabolic stability, demonstrating potential for visualizing and quantifying fear memory.

This study aims to screen the diagnostic biomarkers for fecal antigen of Echinococcus granulosus(E.granulosus)in dogs with high specificity and sensitivity.The sheep-derived EgPSC artificially infected dogs were collected,and the negative and positive fecal samples of dogs with E.granulosus were prepared by arecoline hydrobromide leakage method.Polyclonal antibody,negative fecal antigen-polyclonal antibody conjugates and positive fecal antigen-polyclonal antibody conju-gates were purified by ammonium sulfate precipitation and affinity chromatography,three groups of samples were detected by ELISA and Western blot,LC-MS/MS and bioinformatics analysis were performed on the three groups of samples.The positive fecal antigen-polyclonal antibody con-jugate was used as the treatment group,the polyclonal antibody and the negative fecal antigen-polyclonal antibody conjugates were used as the control groups to screen the unique peptides of the treatment group.ELISA and Western blot showed that only the positive fecal antigen-polyclonal antibody conjugates were positive.According to LC-MS/MS and bioinformatics analysis,11 unique peptides were screened out only in the treatment group.Among them,3 proteins were related to E.granulosus,namely dysferlin,integrator complex 9 and diagnostic antigen gp50,which were mem-brane-associated proteins,INT complex components and diagnostic antigens.This study has pre-liminarily screened out three candidate canine E.granulosus fecal antigen diagnostic markers,pro-viding a reference for further exploration of diagnostic standards for E.granulosus,screening of echinococcosis target genes,and vaccine development.

Objective:To develop and validate a novel PET tracer, N-cyclohexyl-4-((2, 4-dichlorophenyl)(4-(fluoro- 18F)phenyl)methyl)piperazine-1-carboxamide ( 18F-SQKJ-2), targeting cannabinoid type 1 (CB1) receptors for diagnosing psychiatric disorders associated with fear memory. Methods:18F-SQKJ-2 was prepared using a nucleophilic substitution radiochemical synthesis method. For the CB1 receptor blocking experiment, 7 ICR mice were divided into blocking group ( n=4; rimonabant for blocking treatment) and control group 1 ( n=3; no rimonabant blocking treatment). The affinity and specificity of 18F-SQKJ-2 for CB1 receptors were analyzed based on the differences in 18F-SQKJ-2 uptake (percentage injected dose per gram of tissue, %ID/g) by various organs between two groups. The metabolic stability of 18F-SQKJ-2 in vitro was studied using animal tissue homogenates. Ten C57 mice were used to establish fear memory mouse models (fear group, n=6; control group 2, n=4), and the percentage of freezing time was compared between 2 groups. MicroPET scans were used to detect the intracranial distribution of 18F-SQKJ-2, and the relative uptake in each brain region compared to total brain uptake was calculated. Statistical analysis was conducted to compare the differences in CB1 receptor relative total brain uptake in fear-related brain regions between 2 groups. Independent-sample t test and Mann-Whitney U test were used to analyze the data. Results:18F-SQKJ-2 was successfully synthesized with a radiochemical purity ≥98.0% and a corrected radioactive yield of (12.3±6.0)%( n=4). In vitro metabolic stability experiments showed that 18F-SQKJ-2 was basically stable in the liver, blood, and brain within 60min. The CB1 receptor blocking experiment demonstrated that the uptake of 18F-SQKJ-2 in the brains of mice in blocking group was significantly lower than that in control group 1 ((0.95±0.28) vs (3.44±1.16) %ID/g; t=-3.57, P=0.023). The percentage of freezing time in fear group was significantly higher than that in control group 2 (43.28%(39.46%, 52.93%) vs 2.74%(1.52%, 4.85%); Z=-2.45, P=0.010). 18F-SQKJ-2 microPET imaging showed that the uptake of 18F-SQKJ-2 in the cerebral cortex of mice in fear group was significantly increased compared with that in control group 2 ((5.83±0.47)% vs (5.00±0.52)%; t=2.42, P=0.046). Conclusion:18F-SQKJ-2 is successfully prepared with acceptable radiochemical purity and metabolic stability, demonstrating potential for visualizing and quantifying fear memory.

Objective To identify the key genes and targeted protection methods affecting the survival of human islets. Methods Using bioinformatics method, the gene expression profile (GSE53454) was selected through screening and comparison from Gene Expression Omnibus(GEO) database. GEO2R tool was employed to screen the differentially expressed gene(DEG) between the human islets exposed (exposure group) and non-exposed (non-exposure group) to interleukin (IL)-1β and interferon (IFN)-γ for 24, 48 and 72 h, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed by DAVID. Protein-protein interaction (PPI) network was constructed by STRING and Cytoscape apps. Results A total of 69 up-regulated DEGs and 2 down-regulated DEGs were identified. GO analysis showed that during the biological process, DEGs were enriched in the aspects of virus defense and inflammatory response. In cellular components, DEGs were significantly enriched in extracellular space, outside plasma membrane and extracellular regions. Regarding molecular functions, DEGs were significantly enriched in chemokine activity and cytokine activity. KEGG analysis revealed that DEGs were mainly enriched in multiple signaling pathways, such as cytokine-cytokine receptor interaction, virus protein-cytokine and cytokine-receptor interaction, etc. Ten key genes (STAT1, CXCL10, IRF1, IL6, CXCL9, CCL5, CXCL11, ISG15, CD274, IFIT3) with high connectivity were selected by STRING analysis, all of which were significantly up-regulated in human islets exposed to IL-1β and IFN-γ. Six genes (STAT1, CXCL10, CXCL9, CXCL11, CCL5, IL6) were screened by KEGG enrichment analysis, mainly in Toll-like receptor signaling pathway. Conclusions STAT1, CXCL10, CXCL9, CXCL11, CCL5 and IL6 are the key genes affecting the survival of human islets, which are mainly enriched in Toll-like receptor signaling pathway and act as important targets for islet protection.

Objective To evaluate the effect of mesenchymal stem cell (MSC) coated-islets on instant blood-mediated inflammatory reaction (IBMIR) after islet transplantation. Methods MSC labeled with tracer and human islets were placed into an ultra-low adsorption cell culture dish, shaken and mixed twice at an interval of 0.5 h, and then incubated at 37 ℃ and 5% CO₂ for 24 h to obtain MSC-coated islets. The coating effect of MSC and in vitro function of the islets were assessed. A blood circulation tube-shaped model was established in vitro. In the blank control group, 0.2 mL of islet culture solution was added. In the islet group, 800 islet equivalent quantity (IEQ) of uncoated islets were supplemented. In the MSC-coated islets group, 800 IEQ of MSC-coated islets were added, and circulated for 60 min at 37 ℃. A portion of 0.5 mL blood sample was taken for routine blood test at 0, 30 and 60 min, respectively. After 60 min circulation, the blood sample was filtered with a 70 μm filter to collect plasma, blood clots and islets. Blood clots and islets were subject to hematoxylin-eosin (HE) staining and immunohistochemical staining. Morphological changes and the aggregation of CD11b-positive cells surrounding the islets were observed. The contents of plasma thrombin-antithrombin complex (TAT), tissue factor (TF), C3a, C5b-9, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1 and IL-8 were determined by enzyme-linked immune absorbent assay. Results After 24 h co-incubation, the islets were coated by MSC, with a coating degree of approximately 80%. In the islet and MSC-coated islet group, a large quantity of neutrophils and monocytes were observed surrounding the blood clots and islets, and the quantity of CD11b-positive cells in the MSC-coated islet group was less compared with that in the islet group. After co-incubation with the whole blood for 0, 30 and 60 min, the quantity of platelets, neutrophils and monocytes was declined in the MSC-coated and islet groups, and gradually decreased over time. Compared with the blank control group, the quantity of platelets, monocytes and neutrophils was lower, whereas the TF content was higher in the MSC-coated islet group. Compared with the islet group, the quantity of platelets, monocytes and neutrophils was higher, whereas the TAT and TF contents were less in the MSC-coated islet group, the differences were statistically significant (all P < 0.05). Compared with the blank control group, the expression levels of C3a, C5b-9, IL-6, TNF-α and IL-8 were up-regulated in the MSC-coated islet group. Compared with the islet group, the expression levels of C3a, C5b-9, IL-1β, IL-6, TNF-α, IL-8 and MCP-1 were down-regulated in the MSC-coated islet group, and the differences were statistically significant (all P < 0.05). Conclusions MSC-coated islets may reduce the exposure of islet TF in the blood and prevent the incidence of IBMIR during the coagulation response stage, thereby mitigating the injury and loss of islet allograft in the early stage of islet transplantation.

Islet transplantation is one of the effective therapies for diabetes mellitus. Nevertheless, multiple issues still exist, such as shortage of donors and adverse reactions caused by long-term use of immunosuppressants, which limit the islet survival post-transplantation. Microencapsulated islet transplantation may overcome these difficulties to certain extent, whereas many factors, such as the destruction of immune isolation microenvironment within the microcapsules and insufficient supply of oxygen and nutrients, constrain the application of microencapsulated islet transplantation in clinical practice. In recent years, how to enhance the effect of microencapsulated islet transplantation has been gradually studied. The application of stem cells in microencapsulated islet transplantation has steadily become a research hot spot. Therefore, the optimizing strategies for microencapsulated islet transplantation and the application of stem cells in microencapsulated islet transplantation were reviewed, and the potential improvement techniques of microencapsulated islet transplantation were investigated in this article, aiming to provide reference for further clinical application of microencapsulated islet transplantation.

Objective To explore the immunopathological mechanism for the imbalance between the positive signal mediated by inducible costimulator (ICOS) and the negative signal mediated by programmed death-1 (PD-1) in patients with myasthenia gravis (MG).Methods Eighty-two patients with MG,56 healthy controls (HC) and 20 non-MG (NMG) patients,collected in the First Affiliated Hospital of Suzhou University from February 2014 to December 2016,were chosen to participate in the study.The expression of ICOS and PD-1 on peripheral blood mononuclear cells was detected by immuno-fluorescence staining and flow cytometry.The levels of soluble programmed death-1 (sPD-1),soluble programmed death ligand 1 (sPD-L1),IL-4 and other cytokines were detected by enzyme-linked immunosorbent assay.Results (1) Flow cytometry analysis:The co-expression of PD-1,ICOS on CD4 + T cells from MG group (9.64％ (8.82％)) was higher than in HC (1.81％ (2.10％),Z =-7.389,P ＜0.05) and NMG group (2.86％ (1.49％),Z =-4.636,P ＜ 0.05).The expression of ICOS on CD4 + T cells,ICOS ligand (ICOSL) on CD14+ monocytes and CD19+ B cells were increased in MG group comparing with that of the control groups.The proportion of PD-1 + CD4 + T cells (MG group 16.82％ (10.66％),HC 9.34％ (9.18％),Z =-4.345,P＜0.05;NMG group 7.07％ (3.40％),Z=-4.594,P＜0.05) and PD-1 Ligand (PD-L1) + CD14+ monocytes was higher in MG patients.All of these were detected by flow cytometry.(2) ELISA analysis:Serum sPD-1 expression significantly increased in MG group compared with that in the control groups (MG group (1.87 ± 0.64) ng/ml,NMG group (1.49 ± 0.70) ng/ml,t =2.04,P ＜ 0.05;HC (1.05 ± 0.50)ng/ml,t =2.08,P ＜ 0.05),while for serum sPD-L1,there was no significant difference between MG and control groups.(3) Serum cytokines detection:The expression of IL-4 was increased in MG patients (MG group (61.88 ±5.15) pg/ml,HC (32.03 ±1.84) pg/ml,t=2.50,P＜0.05;NMG group (42.62± 3.31) pg/ml,t =2.34,P ＜0.05),and there was a negative correlation between the expression of sPD-1 and the concentration of IL-4.Conclusions The increased expression of PD-1 + ICOS + CD4 + T cells suggested the subset involved in the pathological progress of MG.sPD-1 might disturb the ligation of PD-1 on T cells and PD-L1 on antigen presenting cells,while the ligation of ICOS and ICOSL passed positive signal,leading to over activity of the subsets and the progression of disease.

Objective To discuss the risk factors on short-term prognosis after kidney transplantation from donors after cardiac death (DCD). Methods We retrospectively analyzed the information of donors and recipients who performed DCD donor kidney transplantation in our center between January 2011 and August 2015, including 64 donors and 95 recipients. Also, we analyzed the potential relationship among donors' clinical characteristics and the early recovery of graft function, including the incidence of delayed graft function(DGF)and the serum creatinine (SCr) on the 90th day, and infection rate after kidney transplantation.Results We found that when donors had the factors of WIT>10 min, urine volume<100 ml/h, SBP≤100 mmHg or a history of CPR, the incidence of recipients' DGF were 55.6%,73.3%,62.5%,77.8% respectively with a significant difference. Recipients would have more chance to be infected if donors have the following characteristics: male, older than 50 years, died of cerebral hemorrhage which was caused by cardiovascular diseases, WIT>30 min, treated in ICU for more than 10 days or infection. Conclusions Nowadays, DCD has become the main graft source in Chinese kidney transplantation. This research indicates that the donors' factors may affect the recovery of graft function and the incidence of infection after kidney transplantation to some extent.By evaluating rigorously and preserving quality of renal grafts carefully, DCD would become more safe and valid.

Objective To compare the accuracy of dynamic contrast-enhanced magnetic resonance (DCE-MRI) and SPECT in the measurement of glomerular filtration rate (GFR) in renal allografts.Methods Sixty renal transplant recipients were enrolled in this study.DCE-MRI and SPECT were used to measure the GFR of the transplanted kidneys,and compared with the endogenous creatinine clearance rate (Ccr).Bias,precision,correlation and Bland-Altman agreement were calculated for each modality compared with the endogenous Ccr.Results In 60 renal transplant recipients,the corrected Ccr was (60.63 ± 24.83) ml · min-1 · 1.73 m-2.The GFR measured by SPECT was (65.31 ± 17.08) ml · min-1 · 1.73 m-2,and (50.44 ± 22.78) ml · min-1 · 1.73 m-2 by MRI,respectively.The bias of GFR-SPECT was 4.69 ml·min-1 · 1.73 m-2,and the precision was 23.76 ml·min-1 1.73 m-2.The bias of GFR-MRI was-10.18 ml·min-1 ·1.73 m-2,and the precision was 13.87 ml·min-1 · 1.73 m-2.Correlation analysis showed that GFR-MRI and the endogenous Ccr had a good correlation (r=0.833,P＜0.01),GFR-SPECT and the endogenous Ccr had a moderate correlation (r=0.406,P＜0.01),and GFR-MRI and GFR-MRI had a poor correlation (r=0.342,P ＜0.01).Bland-Altman analysis showed a confidence interval of 95.3 ml·min-1 ·1.73 m-2 for GFR-SPECT and 62.3 ml· min-1 · 1.73 m-2 for GFR-MRI.Conclusion DCE-MRI can be used as confidently as SPECT to evaluate the renal function of transplanted kidneys in the same time of determining anatomical information.