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Aims/Introduction Dipeptidyl peptidase‐4 inhibitor has been proven to improve glycemic control and β‐cell function in latent autoimmune diabetes in adults (LADA). The potential immune modulation mechanism is still unknown. Thus, we tested T‐lymphocyte subsets and expression of relevant transcription factors in LADA patients with sitagliptin intervention for up to 1‐year. Materials and Methods A total of 40 LADA patients were randomly assigned to sitagliptin and/or insulin treatment (SITA group; n = 20) or insulin alone treatment (CONT group; n = 20). Peripheral blood mononuclear cells were isolated at baseline, 6 months and 12 months. The percentage of T‐lymphocyte subsets (T helper 1, T helper 2, T helper 17 and regulatory T cells) tested by flow cytometry, and the messenger ribonucleic acid expression (T box expressed in T cells [T‐BET], GATA binding protein 3 [GATA3], forkhead box protein 3 [FOXP3] and related orphan receptor C [RORC]) tested by real‐time polymerase chain reaction were determined at baseline, 6 months and 12 months. Results The percentage of regulatory T cells in the SITA group was significantly lower than that of the CONT group at baseline. The percentage of T helper 2 cells was higher than that of the CONT group at 6 months and 12 months. At 12 months, the percentage of T helper 17 cells was lower in the SITA group than that of the CONT group. After a 1‐year visit, the messenger ribonucleic acid expression levels of T‐BET expressed in T cells and RORC in the SITA group were significantly lower than at baseline. Whereas that of RORC in the CONT group were significantly lower than that at baseline. Conclusions The data confirmed that sitagliptin altered the phenotype of T cells and downregulated the expression of T‐BET and RORC in LADA patients, and ameliorated glycemic control in LADA patients.

Dipeptidyl peptidase-4 (DPP-4) inhibitors constitute a novel class of anti-diabetic agents confirmed to improve glycemic control and preserve β-cell function in type 2 diabetes. Three major large-scale studies, EXAMINE, SAVOR-TIMI 53, and TECOS, have confirmed the cardiovascular safety profile of DPP-4 inhibitors. Based on these results, DPP-4 inhibitors have gained widespread use in type 2 diabetes treatment. It is currently unknown, however, whether DPP-4 inhibitors have similar therapeutic efficacy against autoimmune diabetes. Several in vitro and in vivo studies have addressed this issue, but the results remain controversial. In this review, we summarize experimental findings and preliminary clinical trial results, and identify potentially effective immune modulation targets of DPP-4 inhibitors for autoimmune diabetes.

The immune system can be divided into adaptive immunity and innate immunity. Adaptive immunity has been confirmed to be involved in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D). However, the role of innate immunity in T1D has only been studied recently. T1D is caused by selective autoimmune destruction of pancreatic islet β cells. A series of studies have suggested that TLRs play a critical role in the pathogenesis of T1D. Aberrant TLR signaling will change immune homeostasis and result in immunopathological conditions such as endotoxin shock and autoimmune responses. Thus, TLR signaling pathways are supposed to be strictly and finely regulated. Epigenetics has recently been proven to be a new regulator of TLR expression. DNA methylation, histone modification, and microRNAs are the three main epigenetic modifications. This review will mainly focus on these epigenetic mechanisms of regulation of TLRs and the role of TLRs in the pathogenesis of T1D.

Chimeric antigen receptor (CAR)-modified T cell therapy has the potential to improve the overall survival of patients with malignancies by enhancing the effectiveness of CAR T cells. Precisely predicting the effectiveness of various CAR T cells represents one of today’s key unsolved problems in immunotherapy. Here, we predict the effectiveness of CAR-modified cells by evaluating the quality of the CAR-mediated immunological synapse (IS) by quantitation of F-actin, clustering of tumor antigen, polarization of lytic granules (LGs), and distribution of key signaling molecules within the IS. Long-term killing capability, but not secretion of conventional cytokines or standard 4-hr cytotoxicity, correlates positively with the quality of the IS in two different CAR T cells that share identical antigen specificity. Xenograft model data confirm that the quality of the IS in vitro correlates positively with performance of CAR-modified immune cells in vivo. Therefore, we propose that the quality of the IS predicts the effectiveness of CAR-modified immune cells, which provides a novel strategy to guide CAR therapy., Graphical Abstract, Xiong et al. developed a novel approach to predict the effectiveness of CAR-modified cells by quantifying the quality of CAR-mediated immunological synapse, which may introduce a new parameter to the field of immunotherapy.

Blockchain-based cryptocurrencies and applications have flourished in blockchain research community. Massive data generated from diverse blockchain systems bring not only huge business values but also technological challenges in data analytics of heterogeneous blockchain data. Different from Bitcoin and Ethereum, EOSIO has richer diversity and a higher volume of blockchain data due to its unique architectural design in resource management, consensus scheme and high throughput. Despite its popularity (e.g., 89,800,000 blocks generated till November 14, 2019 since its launch on June 8, 2018), few studies have been made on data analysis of EOSIO. To fill this gap, we collect and process the up-to-date on-chain data from EOSIO. We name these well-processed EOSIO datasets as XBlock-EOS, which consists of 7 well-processed datasets: 1) Block, Transaction and Action, 2) Internal and External EOS Transfer Action, 3) Contract Information, 4) Contract Invocation, 5) Token Action, 6) Account Creation, 7) Resource Management. It is challenging to process and analyze a high volume of raw EOSIO data and establish the mapping from original raw data to the well-grained datasets since it requires substantial efforts in extracting various types of data as well as sophisticated knowledge on software engineering and data analytics. Meanwhile, we present statistics and exploration on these datasets. Moreover, we also outline the possible research opportunities based on XBlock-EOS., Comment: 15 pages, 12 figures, 7 tables

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8+ T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

A coding variant in PTPN22 (C1858T) is one of the most important genetic risk factors in type 1 diabetes (T1D). The role of the PTPN22 risk allele in B cells is still incompletely understood and has not been investigated directly in T1D. This study aimed to explore the role of PTPN22 in the homeostasis of B cells and its influence in T1D.Wild-type (WT) and Ptpn22 inducible knockdown (KD) NOD mice were treated with 200 μg/ml doxycycline at the age of 10 weeks for 1-2 months. B cell compositions in the bone marrow, peritoneal cavity and spleen were examined. The pathogenicity of Ptpn22 KD B cells was explored by adoptive cell transfer.Ptpn22 silencing increased the frequency of recirculating mature B cells in the bone marrow, decreased the frequency of B-1a cells in the peritoneal cavity and suppressed the formation of marginal zone B cells and plasma cells in the spleen. Changes in the composition of the peripheral B cell compartment caused by altered cell proliferation while rates of apoptosis were not affected. Significantly, co-transfer of Ptpn22 KD B cells with NY8.3 diabetogenic T cells diminished the frequency of diabetes in recipient NOD.scid mice compared with co-transfer of WT B cells.Our study constitutes the first functional study of Ptpn22 in B cells in NOD mice. Our findings suggest that Ptpn22 variation contributes to T1D by modifying the B cell compartment and support a gain-of-function for the PTPN22 disease variant.

Type 1 diabetes (T1D) is an autoimmune disease caused by the dysfunction of immune system and consequently the destruction of insulin-producing β cells. In past decades, numerous studies have uncovered that CD4+ T cell subsets are critical in the pathogenesis of T1D, manifesting that type 1 T helper (Th1) and Th17 cells are pathogenic, while regulatory T (Treg) cells and Th2 cells are protective. More recently, the pathogenic role of another subset, follicular helper T (Tfh) cells that essentially regulate germinal center (GC) formation and humoral responses, has also been demonstrated in T1D and many other autoimmune diseases. In this review, we summarize the evidence for the aberrant differentiation and function of Tfh cells in T1D, and also discuss the underlying mechanisms. A better understanding on the pathogenic role of Tfh cells in T1D will inspire the design of potential therapeutic strategies to target this subset in the future.

With the development of cloud computing, a great amount of information is stored on cloud, such as Facebook, Twitter, and so on. Single cloud or company is not reliable enough for permanent information storage. Once the cloud or company is in downtime, users could lost their information. Multiple clouds can store the information with more security. However, in JointCloud environment, it is a big problem for multiple clouds or companies to maintain the common rights of information for each user. In this paper, we propose EtherShare, a blockchain-based application in JointCloud environment. EtherShare enables users to share information with permanent storage and open access. It keeps the rights of users through smart contracts to make consensus between multiple clouds. And this application is open source on Github.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the immune-mediated destruction of insulin-producing β cells. Recent studies showed that in addition to malaria, artemisinin and its derivative, artesunate (AS), could alleviate several autoimmune diseases. However, whether AS has a role in the prevention or treatment of T1D is still unknown. Therefore, in this study we administrated AS or DMSO in the drinking water of nonobese diabetic (NOD) mice, a mouse model of T1D. We found that AS administration significantly prevented the incidence of T1D. The frequency of IL-4-producing CD4+ single-positive T cells and CD8+ T cells was significantly elevated, and IFN-γ-producing T cells were reduced in the spleen and pancreatic lymph nodes. In the pancreas, the skewing to IL-4-producing T cells was also observed. In addition, more regulatory T cells were found in the pancreas. mRNA levels of proinflammatory cytokines, including TNF-α and IL-6, were decreased. In addition, AS administration promoted the functional maturity of β cells in vitro. Our findings demonstrate that AS administration can prevent T1D in NOD mice mainly by reducing autoimmune T cells and increasing protective T cells. Our data constitute the first functional study of AS in T1D, which may provide a new rationale for future translational studies.-Li, Z., Shi, X., Liu, J., Shao, F., Huang, G., Zhou, Z., Zheng, P. Artesunate prevents type 1 diabetes in NOD mice mainly by inducing protective IL-4-producing T cells and regulatory T cells.

Background: The prediction of mortality for septic acute kidney injury (AKI) has been assessed by a number of potential biomarkers, including long non-coding RNAs (lncRNAs). However, the validation of lncRNAs as biomarkers, particularly for the early stages of septic AKI, is still warranted. Methods: Human blood and urine samples were collected from healthy volunteers(n =30), sepsis patients with AKI (n =96) and sepsis patients without AKI (n=96) for the LncRNA chip assay, RT-qPCR, and absolute real-time PCR analysis. Human renal tubular epithelial (HK-2) cells and C57B6 mice were used to investigate the role and mechanism of LncRNA. and absolute quantification. Findings: Our results indicate that the lncRNA TCONS_00016233 is upregulated in plasma of sepsis-associated non-AKI and AKI patients, but a higher cutoff threshold (9.5x105, copy number) provided a sensitivity of 71.9% and specificity of 89.6% for the detection of AKI. The plasma TCONS_00016233 was highly correlated with serum creatinine, TIMP-2, IGFBP7, and urinary TCONS_00016233. Furthermore, TCONS_00016233 mediates the LPS induced human renal tubular epithelial (HK-2) cells apoptosis. Mechanistically, TCONS_00016233 acts as a ceRNA to prevent miR-22-3p-mediated down-regulation of apoptosis-inducing factor mitochondrion- associated 1(AIFM1). Finally, overexpression of TCONS_00016233 is capable of aggravating the LPS and CLP-induced septic AKI by targeting miR-22-3p/AIFM1 axis. Interpretation: Taken together, our data indicate that TCONS_00016233 may serve as an early diagnosis marker for the septic AKI, possibly acting as a novel therapeutic target for septic AKI. Funding Statement: National Natural Science Foundation of China (81870475, 81570646, 81770951), Hunan Province Natural Science Foundation (2018JJ2568), and excellent Youth Foundation of Hu’nan Scientific Committee (2017JJ1035) supported this study. Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Research protocol was approved by the Second Xiangya Hospital Review Board, The People’s Republic of China Hospital. Written informed consent was got from all individual participants. Animal experiments were performed in strict accordance with the recommendations of the Institutional Committee for the Care and Use of Laboratory Animals of Second Xiangya Hospital (China).

With the rapid development of economy in China, the pharmaceutical industry rises quickly. Although China’s higher medical vocational education develops fast, there are many problems in the current traditional teaching mode and curriculum system, which cannot meet the needs of students. How to reform the education mode under the background of informatization is becoming the focus of vocational education reform in recent years. In this paper, we discussed the teaching effectiveness of DQP framework, an international advanced talent training system of higher vocational education. We found that by choosing reasonable integration of teaching content, different teaching methods, it is in line with the requirements of the times. It facilitates the establishment of pharmacology curriculum standards, the organization of the compilation of pharmacology textbooks which contains the latest knowledge, the use of student-centered flipped classroom teaching mode to improve students’ autonomous learning ability, etc..

Background The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315/IL-10). Methods Female NOD mice at the age of 3–4 weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315/IL-10 (n = 32). The incidence of diabetes was monitored up to 30 weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined. Results Administration with SGAD65190-315/IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315/IL-10 on diabetes and insulitis. Moreover, depletion of CD4+CD25+ T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315/IL-10 vaccination on adoptive transfer of diabetes. Conclusions Our data suggested that SGAD65190-315/IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John Wiley & Sons, Ltd.

Objective: This study aimed to investigate the prevalence of the newly-identified islet autoantibody, tetraspanin 7 autoantibody (TSPAN7A) in Chinese type 1 diabetes (T1D) and Latent Autoimmune Diabetes in Adults (LADA), and the correlation between TSPAN7A and islet function in LADA patients. Research Design and Methods: Patients with newly onset T1D (n=158), long-lasting T1D (n=157), LADA (n=175), type 2 diabetes (T2D; n=204) and healthy control subjects (n=170) were recruited for the cross sectional study. Another cohort including 46 LADA patients with 3-year follow-up was recruited forthe longitudinal study. TSPAN7A was assayed by luciferase immunoprecipitation system assay. Results: The prevalence of TSPAN7A in newly onset T1D, non-newly onset T1D, T2D, LADA and healthy control subjects were 25.3% (40/158), 10.2% (16/157), 0.5% (1/204), 21.1 (37/175) and 1.2% (2/170), respectively. Out of the 23 patients with newly onset T1D but negative for GADA, IA2 and ZnT8A, 3 patients were positive for TSPAN7A (13.0%) improving the detection of autoantibodies from 81.6% to 83.5% in T1D. In LADA patients, analysis of logistic regression demonstrated that duration (OR=1.77, P=0.026), GADA titer (OR=2.79, P=0.006) and TSPAN7A (OR=2.86, P=0.034) were the risk factors to islet function while BMI was the protection factor (OR=0.35, P=0.001). In the follow-up study, LADA patients with TSPAN7A showed a worse islet function compared those negative for TSPAN7A after 3-year diagnosis. Conclusion: TSPAN7A is a novel diagnostic tool for T1D and LADA. Combination of TSPAN7A with other autoantibodies can improve the diagnosis of T1D. TSPAN7A is negatively correlated with islet function in LADA. Disclosure Z. Zhou: None. X. Shi: None. P. Zheng: None. G. Huang: None. Y. Wang: None. C. Deng: None.

Blockchain systems, with the characteristics of decentralization, irreversibility and traceability, have attracted a lot of attentions recently. However, the current performance of blockchain is poor, which becomes a major constraint of its applications. Additionally, different blockchain systems lack standard performance monitoring approach which can automatically adapt to different systems and provide detailed and real-time performance information. To solve this problem, we propose overall performance metrics and detailed performance metrics for the users to know the exact performance in different stages of the blockchain. Then we propose a performance monitoring framework with a log-based method. It has advantages of lower overhead, more details, and better scalability than the previous performance monitoring approaches. Finally we implement the framework to monitor four well-known blockchain systems, using a set of 1,000 open-source smart contracts. The experimental results show that our framework can make detailed and real-time performance monitoring of blockchain systems. We also provide some suggestions for the future development of blockchain systems.

Blockchain technology becomes increasingly popular. It also attracts scams, for example, Ponzi scheme, a classic fraud, has been found making a notable amount of money on Blockchain, which has a very negative impact. To help dealing with this issue, this paper proposes an approach to detect Ponzi schemes on blockchain by using data mining and machine learning methods. By verifying smart contracts on Ethereum, we first extract features from user accounts and operation codes of the smart contracts and then build a classification model to detect latent Ponzi schemes implemented as smart contracts. The experimental results show that the proposed approach can achieve high accuracy for practical use. More importantly, the approach can be used to detect Ponzi schemes even at the moment of its creation. By using the proposed approach, we estimate that there are more than 400 Ponzi schemes running on Ethereum. Based on these results, we propose to build a uniform platform to evaluate and monitor every created smart contract for early warning of scams. STINT Blockchain

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell–cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically “stacked” cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.

Background DiGeorge syndrome affects more than 3.5 million persons worldwide. Partial DiGeorge syndrome (pDGS), which is characterized by a number of gene deletions in chromosome 22, including the chicken tumor virus number 10 regulator of kinase (Crk)–like (CrkL) gene, is one of the most common genetic disorders in human subjects. To date, the role of natural killer (NK) cells in patients with pDGS remains unclear. Objective We sought to define the effect of pDGS-related Crk haploinsufficiency on NK cell activation and cytotoxic immunological synapse (IS) structure and function. Methods Inducible CrkL-silenced NK cells were used to recapitulate the pDGS, CrkL-haploinsufficient phenotype. Findings were validated by using NK cells from patients with actual pDGS. Ultimately, deficits in the function of NK cells from patients with pDGS were restored by lentiviral transduction of CrkL. Results Silencing of CrkL expression inhibits NK cell function. Specifically, pDGS haploinsufficiency of CrkL inhibits accumulation of activating receptors, polarization of cytolytic machinery and key signaling molecules, and activation of β 2 -integrin at the IS. Reintroduction of CrkL protein restores NK cell cytotoxicity. Conclusion CrkL haploinsufficiency causes functional NK deficits in patients with pDGS by disrupting both β 2 -integrin activation and activating receptor accumulation at the IS. Our results suggest that NK cell IS quality can directly affect immune status, providing a potential target for diagnosis and therapeutic manipulation in patients with pDGS and in other patients with functional NK cell deficiencies.

PTPN22 encodes the lymphoid tyrosine phosphatase (LYP) and is the second strongest non-HLA genetic risk factor for type 1 diabetes. The PTPN22 susceptibility allele generates an LYP variant with an arginine-to-tryptophan substitution at position 620 (R620W) that has been reported by several studies to impart a gain of function. However, a recent report investigating both human cells and a knockin mouse model containing the R620W homolog suggested that this variation causes faster protein degradation. Whether LYP R620W is a gain- or loss-of-function variant, therefore, remains controversial. To address this issue, we generated transgenic NOD mice (nonobese diabetic) in which Ptpn22 can be inducibly silenced by RNA interference. We found that Ptpn22 silencing in the NOD model replicated many of the phenotypes observed in C57BL/6 Ptpn22 knockout mice, including an increase in regulatory T cells. Notably, loss of Ptpn22 led to phenotypic changes in B cells opposite to those reported for the human susceptibility allele. Furthermore, Ptpn22 knockdown did not increase the risk of autoimmune diabetes but, rather, conferred protection from disease. Overall, to our knowledge, this is the first functional study of Ptpn22 within a model of type 1 diabetes, and the data do not support a loss of function for the PTPN22 disease variant.

Context: The discrepancies in terms of human leukocyte antigen (HLA)-DRB1-DQA1-DQB1 conferred risks between latent autoimmune diabetes in adults (LADA) and type 1 diabetes (T1D) patients remained almost completely unknown. The goal of the current study is to determine and compare HLA-conferred risks between LADA and T1D. Design: A case-control study was conducted in a representative Chinese data set containing 520 T1D patients, 562 LADA patients, and 1065 controls. The frequencies and odds ratios for HLA susceptible haplotypes and genotypes and for arginine at residue 52 in the DQ-α chain or aspartic acid at residue 57 in the DQ-β chain were analyzed. Results: DRB1*0405-DQA1*03-DQB1*0401 and DRB1*0901-DQA1*03-DQB1*0303 are the major LADA susceptible haplotypes, which also confer comparable risks for T1D (odds ratio 2.02 vs 2.20 and 1.61 vs 2.30, respectively). The strongly associated T1D haplotype DRB1*0301-DQA1*05-DQB1*0201 is also associated with LADA but confers only half of the T1D risk (odds ratio 2.65 vs 4.84). Interestingly, the most susceptible T1D haplotypes, DRB1*0901-DQA1*05-DQB1*0201, DRB1*0301-DQA1*03-DQB1*0201, and DRB1*0301-DQA1*03-DQB1*0303, are not associated with LADA. Genotypes for DR3/DR3, DR3/DR9, and DR9/DR9 are highly associated with T1D susceptibility, whereas only DR9/DR9 confers risk for LADA. DR3/DR3 is the high-risk genotype in Chinese T1D patients, which manifests similar risk as the DR3/DR4 genotype in Caucasians but with a lower frequency. DR9/DR9 is the high risk LADA genotype in Chinese. Alleles with DQ-α arginine at residue 52-positive, DQ-β aspartic acid at residue 57-negative, and their combination formed in cis or trans confer susceptibility to T1D but not to LADA. Conclusion: Our results suggest that LADA risk conferred by HLA-DRB1-DQA1-DQB1 loci in Chinese differs significantly from that of T1D risk. This information would be useful for classifying Asian LADA patients, which should provides novel insight into the understanding of its pathoetiology as well.

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated CrusEs2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CrusEs2 was of 1237 bp, containing a 5′ untranslated region (UTR) of 12 bp, a 3′ UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The CrusEs2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that CrusEs2 should be classified as a type III crustin. The mRNA transcripts of CrusEs2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of CrusEs2 was cloned into pET-21a (+) and expressed in Escherichia coli. The purified recombinant CrusEs2 inhibited the growth of Gram-positive bacteria at MIC of 0.093–0.37 μM. The results indicated that CrusEs2 was involved in immune response of E. sinensis against bacterial challenge.

OBJECTIVE CTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. In this study, we investigated the contribution of sCTLA-4 to immune regulation with the aim to elucidate the functional basis of the disease association of CTLA4. RESEARCH DESIGN AND METHODS To model the disease-associated splicing variation of CTLA4, we generated NOD mice in which sCTLA-4 mRNA is silenced by RNA interference. RESULTS We found that loss of sCTLA-4 impairs the function of regulatory T (Treg) cells. This functional defect could be attributed, at least in part, to the failure of sCTLA-4 knockdown (KD) Treg cells to downregulate dendritic cell costimulation. sCTLA-4 KD Treg cells, in contrast with wild-type Treg cells, failed to inhibit colitis induced by transfer of CD4+CD45RBhi cells into NOD.SCID animals. Furthermore, diminished sCTLA-4 expression accelerated the onset of autoimmune diabetes in transgenic mice. CONCLUSIONS Our results demonstrate that sCTLA-4 participates in immune regulation by potentiating the function of Treg cells. The functional outcome of silencing this splice variant in the NOD model provides an explanation for the association of CTLA4 variation with autoimmunity. Lower sCTLA-4 expression from the susceptibility allele may directly affect the suppressive capacity of Treg cells and thereby modulate disease risk. Our unprecedented approach establishes the feasibility of modeling splicing variations relevant to autoimmunity.

Background Recent discoveries from animal models demonstrated that neutrophils can induce type 1 diabetes (T1D) through infiltrating into the islets. However, the evidence of their actions in T1D patients is relatively rare, and the change trend of neutrophil numbers and functions in different subtypes of diabetes has not been investigated. Methods Patients with newly diagnosed T1D (n = 189), latent autoimmune diabetes in adults (LADA) (n = 86), T2D (n = 235), and healthy controls (n = 709) were enrolled. Circulating neutrophil counts were measured, and their correlations with clinical parameters were analysed. Neutrophils were isolated by density gradient centrifugation and magnetic bead cell sorting method. Neutrophil migration rate and chemokine levels in the blood were explored by trans-well and ELISA, respectively. Neutrophil phagocytosis rate, adhesion molecules and chemokine receptors expression were investigated by flow cytometry. Results Compared with controls, neutrophil counts decreased in T1D patients but increased in T2D patients, with no change in LADA patients. The numbers showed a gradual increase trend from T1D, LADA to T2D. In autoimmune diabetes, neutrophil counts were associated with the number and titre of positive autoantibodies against β-cell antigens. No difference was found in neutrophil phagocytosis rate, but neutrophil migration in T1D patients was impaired and associated with CD62L expression, which was related closely to the titre of autoantibody. Conclusions Neutrophil numbers and migration abilities displayed distinct levels in different types of diabetes. In T1D, CD62L seems to play an important role in the migration of neutrophils and β-cell autoimmunity.

Summary Type 1 diabetes (T1D) is an autoimmune disease, which is characterized by the destruction of islet β cells in the pancreas triggered by genetic and environmental factors. In past decades, extensive familial and genome‐wide association studies have revealed more than 50 risk loci in the genome. However, genetic susceptibility cannot explain the increased incidence of T1D worldwide, which is very likely attributed by the growing impact of environmental factors, especially gut microbiome. Recently, the role of gut microbiome in the pathogenesis of T1D has been uncovered by the increasing evidence from both human subjects and animal models, strongly indicating that gut microbiome might be a pivotal hub of T1D‐triggering factors, especially environmental factors. In this review, we summarize the current aetiological and mechanism studies of gut microbiome in T1D. A better understanding of the role of gut microbiome in T1D may provide us with powerful prognostic and therapeutic tools in the near future.

NF-κB is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-κB-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor κB (IκB)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-κBs and phylogenetic analysis suggested that EsRelish was a member of the NF-κB family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

Latent autoimmune diabetes in adults (LADA) is a form of autoimmune diabetes with heterogeneous features. This study aimed to investigate the persistent status of glutamic acid decarboxylase antibody (GADA) in patients with LADA and its association with clinical characteristics.This 3-year follow-up study enrolled 107 LADA and 40 type 2 diabetes mellitus (T2DM) patients from October 2005 to December 2013. GADA titer, epitopes, and clinical characteristics (including fasting C-peptide and HbA1c ) in LADA patients were assayed annually. The human leukocyte antigen DQ (HLA-DQ) genotypes were also analysed. The relationship between the persistence of GADA and the clinical characteristics was investigated in LADA patients.After 3-year follow-up, 36.5% (39/107) LADA patients remained GADA positive (persistently positive group), 19.6% (21/107) patients fluctuated positively and negatively (fluctuating group), and 43.9% (47/107) patients became GADA negative, among which 61.7% (29/47) seroconversions occurred within 6 months of follow-up (transiently positive group). The GADA persistently positive group possessed higher titer of GADA than transiently positive group and fluctuant group (all p = 0.000), higher reactivities to middle and C-terminal regions of GAD65 than those in transiently positive group (p = 0.001 and p = 0.000, respectively), and lower baseline fasting C-peptide level than T2DM patients and transiently positive group [415(31-1862) vs 620(220-1658) pmol/L, p = 0.014; and 415(31-1862) vs 705(64-1541) pmol/L, p = 0.017, respectively]. The GADA transiently positive group retained a higher HbA1c level when compared with T2DM patients (p = 0.023). In addition, the three LADA groups shared similar frequencies of HLA-DQ susceptible haplotypes that were higher as compared with T2DM. The GADA persistently positive group had a higher annual declining rate in fasting C-peptide than T2DM patients [-14%(-174-33%) vs -1%(-27-28%), p = 0.007].The LADA patients with GADA transient positivity account for a large proportion, whose clinical characteristics and HLA-DQ haplotypes are different from those of T2DM. The patients with high titer GADA and reactivities to GADA65 middle and C-terminal regions showed a persistent GADA positivity, in which a worse baseline and accelerated decline of β-cell function need early intervention in the practice. Copyright © 2016 John WileySons, Ltd.

The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315 /IL-10).Female NOD mice at the age of 3-4 weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315 /IL-10 (n = 32). The incidence of diabetes was monitored up to 30 weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined.Administration with SGAD65190-315 /IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315 /IL-10 on diabetes and insulitis. Moreover, depletion of CD4(+) CD25(+) T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315 /IL-10 vaccination on adoptive transfer of diabetes.Our data suggested that SGAD65190-315 /IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John WileySons, Ltd.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

Crustin is a cysteine-rich antibacterial peptide which widely distributes within decapod crustaceans. In the present study, the cDNA encoding crustin-like peptide (designated CrusEs) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag analysis. The full-length cDNA of CrusEs was of 796 bp, containing a 5' untranslated region (UTR) of 214 bp, a 3' UTR of 267 bp with a poly(A) tail, and an open reading frame (ORF) of 315 bp encoding a polypeptide of 104 amino acids including a putative signal peptide of 21 amino acids. A WAP domain and the consensus framework existing in class I crustins were both identified in CrusEs, suggesting that CrusEs is a new member of type I crustins. Quantitative real-time RT-PCR was employed to examine the expression of CrusEs, and its mRNA transcript was mainly detected in haemocytes and gill. The CrusEs mRNA transcript in haemocytes was down-regulated after the challenge with Gram-positive bacteria Micrococcus luteus, while Gram-negative bacteria Listonella anguillarum did not induce significant variation of CrusEs mRNA. The mature peptide of CrusEs was cloned into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant CrusEs inhibited the growth of Gram-positive bacteria with low MIC. The results indicate that CrusEs is a potent antibacterial protein against Gram-positive bacteria infection, and it may play an important role in innate immune response of E. sinensis. (C) 2010 Elsevier Ltd. All rights reserved.

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds, interacts with a variety of proteins and therefore regulates their activities. One CypA (designated CfCypA) cDNA was cloned from Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CfCypA consisted of 1,248 nucleotides with a canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an open reading frame (ORF) of 495 nucleotides encoding a polypeptide of 164 amino acids. The deduced amino acid sequence shared high similarity with CypA from the other species, indicating that CfCypA should be a new member of the CypA family. Quantitative real-time (RT) PCR was employed to assess the mRNA expression of CfCypA in various tissues and its temporal expression in haemocytes and gonad of scallops challenged with Vibrio anguillarum. The mRNA transcripts of CfCypA could be detected in all the examined tissues with highest expression level in gonad. After bacterial challenge, the expression level of CfCypA was almost unchanged in haemocytes, but up-regulated in gonad and increased to the peak (22.59-fold; P < 0.05) at 4 h post-injection, and then dropped to the original level at 8 h post-injection. These results indicated that CfCypA was constitutive expressed in haemocytes, but could be induced in gonad, and perhaps played a critical role in response to the bacterial challenge in gonad.

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

The ligation of programmed cell death-1 (PD-1) to its ligands PD-L1 and PD-L2 counteracts T-cell activation, which is critical in immune tolerance. The persistent high expression of PD-1 and PD-L1 are also observed on tumor-infiltrating lymphocytes and various tumor cells, maintaining the highly suppressive microenvironment in tumor sites and promoting tumor malignancies. The blockade of PD-1 axis with PD-L2 fusion protein or monoclonal antibodies against either PD-1 or PD-L1 has been clinically evaluated in various tumor types. This short review summarizes the progress of PD-1 axis blockade in clinical trials to evaluate its effectiveness in the antitumor immunotherapy.

Very few studies have addressed how CrkL, a ubiquitous adaptor protein, regulates the immune response, especially in NK cells. Previous studies indicate that CrkL is required for NKG2D-mediated cellular cytotoxicity (NC) and CD16 signaling triggered antibody mediated cellular cytotoxicity (ADCC). However, how CrkL protein controls NK cell cytotoxicity remains unclear. Here we used lentiviral inducible shRNA to silence CrkL in human NK cell line and primary NK cells. Inducible knockdown of CrkL significantly reduced NK-target conjugation, degranulation, and cytotoxicity in both NC- and ADCC-mediated immune responses. Further investigation on the function and signaling transduction at the NK cytotoxic immunological synapse (IS) reveals that polarization of lytic granules, and accumulation of both F-actin and phosphorylated Vav-1 at the IS were greatly reduced in CrkL-silenced NK cells. NK cell activation involves in actin-dependent activating receptor clustering. CrkL silencing resulted in reduced accumulation of ligands for CD16 and NKG2D on the lipid bilayer. Similar results were obtained in NK cells from DiGeorge Syndrome patients (CrkL gene deletion). We thus propose a new model for adaptor protein CrkL function in NK cell activation: CrkL controls NK cell cytotoxicity via promoting F-actin accumulation and lytic granule polarization by modulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at the IS.

Type 1 diabetes is a polygenic autoimmune disorder. One of the genetic polymorphisms linked to type 1 diabetes, as well as to other autoimmune diseases, is located within the CTLA-4 gene. This polymorphism is associated with a change in splicing frequency, altering the ratio between full-length and soluble CTLA-4 (sCTLA-4) in human. The role and function of sCTLA-4 are as yet unknown. To investigate the sCTLA-4 isoform in a model organism, we generated nonobese diabetic (NOD) mice in which expression of sCTLA-4, but not full-length or ligand-independent CTLA-4, is constitutively reduced by RNAi. We find that loss of sCTLA-4 significantly reduces the potency of regulatory T cells, providing a possible explanation for the effect of the human CTLA-4 polymorphism on disease susceptibility. Our study demonstrates the utility of RNAi for the study of single splice variants in vivo, and supports a role for sCTLA-4 in regulatory T cell function.