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HBV-related acute-on-chronic liver failure (HBV-ACLF) is the most common type of liver failure with high mortality. Artificial liver support system (ALSS) is an important mean to reduce the mortality of HBV-ACLF but lacking index to assess its effectiveness. The cytokines are closely related to the prognosis of HBV-ACLF patients with ALSS treatment, however, which is not fully understood.One hundred forty-two patients with HBV-ACLF and 25 healthy donors were enrolled. The cytokine profile of peripheral blood was determined in the patients before and after ALSS treatment, and their relationship with effectiveness of ALSS treatment in HBV-ACLF was analyzed.Serum IL-28A levels were markedly lower in ALSS-effective patients than those in non-effective patients pre-ALSS treatment. Similarly, serum IL-6 was significantly lower in ALSS-effective patients. Furthermore, for patients with effective treatment, serum IL-28A levels were positively related with IL-6 levels post-ALSS (r = 0.2413, p = 0.0383). The ROC curve analysis showed that serum levels of IL-28A (AUC = 0.6959 when alone or 0.8795 when combined with total bilirubin, platelet count and INR, both p 0.0001) and IL-6 (AUC = 0.6704, p = 0.0005) were useful indices for separating effective from non-effective ALSS treatment of HBV-ACLF patients. Multivariate logistic regression analysis demonstrated that lower level of IL-28A was independently associated with higher effective rate of ALSS treatments.Lower level of IL-28A is a predictive biomarker for ALSS in effective treatment of HBV-ACLF patients and IL-28A may be potential target for the treatment of HBV-ACLF.

The human microbiome has attracted attention for its potential utility in precision medicine. Increasingly, more researchers are recognizing changes in intestinal microbiome can upset the balance between pro- and anti-inflammatory factors of host immune system, potentially contributing to arthritis immunopathogenesis. Patients who develop rheumatoid arthritis from undifferentiated arthritis can face multiple irreversible joint lesions and even deformities. Strategies for identifying undifferentiated arthritis patients who have a tendency to develop rheumatoid arthritis and interventions to prevent rheumatoid arthritis development are urgently needed. Intestinal microbiome dysbiosis and shifts in the miRNA profile affect undifferentiated arthritis progression, and may play an important role in rheumatoid arthritis pathophysiologic process via stimulating inflammatory cytokines and disturbing host and microbial metabolic functions. However, a causal relationship between microbiome–miRNA interactions and rheumatoid arthritis development from undifferentiated arthritis has not been uncovered yet. Changes in the intestinal microbiome and miRNA profiles of undifferentiated arthritis patients with different disease outcomes should be studied together to uncover the role of the intestinal microbiome in rheumatoid arthritis development and to identify potential prognostic indicators of rheumatoid arthritis in undifferentiated arthritis patients. Herein, we discuss the possibility of microbiome–miRNA interactions contributing to rheumatoid arthritis development and describe the gaps in knowledge regarding their influence on undifferentiated arthritis prognosis that should be addressed by future studies.

Background T cells play an important role in the prognosis of hepatitis B virus (HBV) infection, and are involved in the seroconversion of a patient from HBsAb negative to positive. To compare the T-cell receptor β-chain variable region (TcRBV) complementarity-determining region 3 (CDR3) in subjects with or without hepatitis B surface antigen (HBsAg) convert to hepatitis B surface antibody (HBsAb), the TcRBV was determined using high throughput sequencing (HTS). Methods The clonotype and diversity of CDR3 in peripheral blood mononuclear cells of subjects with resolved acute hepatitis B (AHB, HBsAb+, HBsAg-) (n = 5), chronic hepatitis B (CHB, HBsAb-, HBsAg+) (n = 5), and healthy controls (HC, HBsAb-, HBsAg-) (n = 3) were determined and analyzed using HTS (MiSeq). Results The overlapping rate of CDR3 clones of any two samples in AHB group was 2.00% (1.74% ~ 2.30%), CHB group was 1.77% (1.43% ~ 2.61%), and HC group was 1.82% (1.62% ~ 2.12%), and there was no significant difference among the three groups by Kruskal-Wallis H test. However, among the top 10 cumulative frequencies of clonotypes, only the frequency of clonotype (TcRBV20–1/BD1/BJ1–2) in AHB group was lower than that of HC group (P P > 0.05). Conclusions Thus, there are 57 TcRBV clonotypes that may be related to HBsAg seroconversion of AHB subjects, but the diversity of TcRBV CDR3 is not significantly related to the HBsAb positive status.

Purpose To identify candidate hub genes and miRNAs associated with active tuberculosis (ATB) and reveal the potential molecular mechanisms of disease progression. Patients and methods The expression of mRNA and miRNA was evaluated in peripheral blood mononuclear cells (PBMC) from 4 ATB patients and 4 healthy donors (HD) using high throughput sequencing (HTS) and bioinformatics analysis. Moreover, differentially expressed miRNAs were validated with 35 ATB patients and 35 HDs using reverse transcription quantitative real-time PCR (RT-qPCR). Results A total of 2658 significantly differentially expressed genes (DEG) including 1415 up-regulated genes and 1243 down-regulated genes were identified in the ATB group compared with HDs, and the DEGs enriched in immune-related pathways, especially in TNF signaling pathway, cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathways and tuberculosis. Additionally, 10 hub genes were acquired according to protein-protein interaction (PPI) analysis of DEGs. Moreover, 26 differentially expressed miRNAs were found in ATB group compared with HDs. Furthermore, RT-qPCR results showed that hsa-miR-23a-5p (P=0.0106), hsa-miR-183-5p (P=0.0027), hsa-miR-193a-5p (P=0.0021) and hsa-miR-941(P=0.0001) were significantly increased in the ATB patients compared with HD group, and the hsa-miR-16-1-3p was significantly decreased (P=0.0032). Conclusion Our research provided a characteristic profile of mRNAs and miRNAs expressed in ATB subjects, and 10 hub genes related with ATB were found, which will contribute to explore the role of miRNAs and hub genes in the pathogenesis of ATB, and improve the ability of differential diagnosis and treatment for the disease.

To study the homogeneity and heterogeneity of CD4+CD25+ T cells receptor β-chain complementarity determining region 3 (TCR β CDR3) repertoires in breast tumor tissues, lung metastatic tissues, and spleens from 4T1 tumor-bearing BALB/c mice. We used high-throughput sequencing to analyze the characteristics and changes of CD4+CD25+ TCR β CDR3 repertoires among tumor tissues, lung metastatic tissues, and spleens. The diversity of the CD4+CD25+ TCR β CDR3 repertoires in breast tumor tissue was similar to that of lung metastatic tissues and less pronounced than that of spleen tissues. Breast tumor tissues and lung metastatic tissues had a greater number of high-frequency CDR3 sequences and intermediate-frequency CDR3 sequences than those of spleens. The proportion of unique productive CDR3 sequences in breast tumor tissues and lung metastatic tissues was significantly greater than that in the spleens. The diversity and frequency of the CDR3 repertoires remained homogeneous in breast tumors and lung metastatic tissues and showed great heterogeneity in the spleens, which suggested that the breast tissues and lung metastatic tissues have characteristics of CD4+CD25+ T cells that relate to the tumor microenvironment. However, the number and characteristics of overlapping CDR3 sequences suggested that there were some different CD4+CD25+ T cells in tumors and in the circulatory immune system. The study may be used to further explore the characteristics of the CDR3 repertoires and determine the source of the CD4+CD25+ T cells in the breast cancer microenvironment.

Jiezuan Yang,1 Yongtao Li,1 Jing Ye,2 Ju Wang,1 Haifeng Lu,1 Xinsheng Yao3 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Infectious Diseases, Hangzhou, People’s Republic of China; 2Department of Surgical ICU, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, People’s Republic of China; 3Department of Immunology, Research Center for Medicine & Biology, Innovation & Practice Base for Graduate Students Education, Zunyi Medical University, Zunyi, People’s Republic of ChinaCorrespondence: Jiezuan Yang, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Infectious Diseases, 79 Qingchun Road, Hangzhou, 310003, People’s Republic of China, Tel +86 571 87236756, Email yangyan@zju.edu.cnBackground: Hepatitis B (HepB) vaccination can effectively prevent the prevalence of hepatitis B virus (HBV) infection. However, the incidence of vaccination failure is about 5∼ 10% and the underlying molecular mechanisms are poorly understood. T cells have an essential role in the recipient’s immune response to vaccine, which could be elucidated by high-throughput sequencing (HTS) and bioinformatics analysis.Methods: We conducted HTS of the T cell receptor β chain (TRB) complementarity-determining region 3 (CDR3) repertoires in eighteen positive responders (responders) and 10 negative responders (non-responders) who all had HepB vaccination, the repertoire features of BV, BJ and V-J genes and their diversity, respectively, were compared between the positive and negative responders using the Mann–Whitney test. Moreover, the relatively conserved motifs in CDR3 were revealed and compared to those in the other group’s report.Results: The diversity of TRB CDR3 and the frequencies of BV27 and BV7-9 are significantly increased for HepB vaccine responders compared to those in non-responders. The motifs of CDR3s in BV27/J1-1, BV27/J2-5, and BV7-9/J2-5, respectively, were most expressed as “NTE”, “QETQ”, and “GG-Q (E)-ETQ”. Moreover, the motif “KLNSPL” was determined in nearly 80% CDR3s in BV27/J1-6 from HepB vaccine responders for the first time.Conclusion: Our results present the comprehensive profiles of TRB CDR3 in the HepB vaccine responders and non-responders after standard vaccination protocol and determine the relatively conservative motifs of CDR3s that may respond to the HepB vaccine. Further results suggest that the profile of TRB repertoire could distinguish the HepB vaccine responders from non-responders and provide a new target for optimizing and improving the efficiency of the HepB vaccine.Keywords: hepatitis B vaccination, hepatitis B surface antibody, complementarity-determining region 3, immune repertoire, high-throughput sequencing

BACKGROUND T cells with edited T cell receptor β-chain variable (TRBV) are involved in the immune response to recombinant hepatitis B surface antigen (rHBsAg) vaccine and the production of hepatitis B surface antibody (HBsAb). The immune repertoire (IR) profile and mechanism of vaccination positive responders (VPR) with rHBsAg are not fully understood. METHODS The IR of six VPRs (HBsAb+, HbsAg-) with rHBsAg vaccination was established by the high throughput sequencing technique and bioinformatics analysis and compared with those in five vaccination negative responders (VNRs) (HbsAb-, HbsAg-) who were also inoculated with rHBsAg. The repertoire features of the BV, BJ and V (CDR3) J genes and immune diversity in peripheral blood mononuclear cells, respectively, were analyzed for each subject. RESULTS There was no significant difference in sequencing amplification indices of each sample. However, TRBV15/BJ2-3 demonstrated significantly high expression levels in VPR compared to those in the VNR group (both p

Background: Although gut microbiota dysbiosis has recently been reported in HIV infected individuals, the relationship between the gut microbiota and immune activation in patients with a different immune response to highly active antiretroviral therapy (HAART) is still not well understood. Gut microbiota and immune activation were studied in 36 non-HIV-infected subjects (Health Control) and 58 HIV-infected individuals which included 28 immunological responders (IR) and 30 immunological non-responders (INR) (≥500 and Fusobacterium, Ruminococcus_gnavus and Megamonas were more abundant, whereas Faecalibacterium, Alistipes, Bifidobacterium, Eubacteriumrectale and Roseburia were more depleted in the IR and INR groups than that in the health control. Ruminococcaceae and Alistipes were positively correlated with nadir and current CD4+ T-cell counts, but negatively correlated with CD8+CD57+ T-cell counts. Inflammation markers and translocation biomarkers (LPS) levels were positively correlated with the abundance of genus Ruminococcus and Fusobacterium, but were negatively correlated with the genus Faecalibacterium. The relative abundances of Escherichia-Shigella and Blautia were significantly higher in the IR than that in the INR group. Escherichia-Shigella were negatively correlated with the CD4/CD8 ratio but positively correlated with the amount of CD8+CD57+ T-cells. Roseburia and Blautia were negatively associated with nadir CD4+ T-cell and positively associated with CD8+CD57+ T-cell counts. Conclusions: Gut microbiota dysbiosis may be one of the factors contributing to different immune response to HAART. Fusobacterium, Alistipes, Ruminococcaceae, Faecalibacterium, Escherichia-Shigella, Roseburia and Blautia maybe the major genus contributed to different immune response in immunodiscordant and immunoconcordant patients on long-term suppressive ART. Blautia and Roseburia might be associated with treatment outcome.

The chronicity of Hepatitis B virus (HBV) infection relates to both viral factors and host factors. HBV could result in persistent infection and even serious liver disease, including chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma (HCC). Although the HBV vaccine can effectively prevent HBV infection, chronic HBV infection still endangers human health and results in a large social burden. Moreover, the mechanisms underlying the HBV-mediated imbalance of the immune response and persistent infection are not fully understood. Exosomes are extracellular vesicles (EVs) 40-160 nm in size that are released from many cells and transfer specific functional RNAs, proteins, lipids and viral components from donor to recipient cells. These exosome nanovesicles are associated with various biological processes, such as cellular homeostasis, immune response and cancer progression. Besides, previous studies on exosomes have shown that they take part in viral pathogenicity due to the similarity in structure and function between exosomes and enveloped viruses. Moreover, exosome as a novel immunomodulatory carrier plays a significant role in viral immunology. In this review, we focus on the latest progress in understanding the role of exosomes in HBV transmission as well as their vital roles in immune regulation during HBV infection. Furthermore, we discuss the potential clinical applications of exosomes in hepatitis B infection, including the use of exosomes in the auxiliary diagnosis and treatment of hepatitis B.

The ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) is a key factor involved in coagulation process and plays a vital role in the progression and prognosis of chronic hepatitis B (CHB) patients with antiviral treatment. However, there are few reports about the profile of plasma ADAMTS13 in CHB patients during entecavir maleate (m-ETV) treatment. One hundred two HBV e antigen (HBeAg)–positive CHB patients on continuous m-ETV naive for at least 96 weeks were recruited. Patients with liver cirrhosis were excluded using liver biopsies and real-time elastography. Plasma ADAMTS13 and interleukin 12 (IL-12) levels were evaluated at baseline and12, 24, 48, 72, and 96 weeks, respectively. The change of ADAMTS13 (ΔADAMTS13) and IL-12 (ΔIL-12) possesses a significant relationship in CHB patients with HBeAg seroconversion (SC) at 48-week m-ETV treatment (p < 0.001), but no significance in patients without SC. Furthermore, Cox multivariate analysis demonstrated that the change of ADAMTS13 (IL-12) is an independent predictor for HBeAg SC at week 96, and the area under the receiver operating characteristic curve for the ΔADAMTS13 (ΔIL-12) in CHB patients with 48-week m- ETV treatment is 0.8204 (0.8354) (p < 0.001, both) to predict HBeAg SC at week 96. The results suggested that higher increased ADAMTS13 and IL-12 after 48-week m-ETV treatment contributed to an enhanced probability of HBeAg SC, although the mechanism is undetermined. Quantification of ADAMTS13 (IL-12) during m-ETV treatment may help to predict long-term HBeAg SC in CHB patients.

Liver biopsy is the current reliable way of evaluating liver fibrosis. However, no specific sera biomarker could be applied in clinical diagnosis. As the pivotal role of osteopontin (OPN) reported in numerous liver diseases, thrombin‐cleaved OPN (Thr‐OPN) exposes an integrin‐binding motif that promoted biological functions. Herein, we investigated the potential of Thr‐OPN in liver fibrosis. Using patient samples, mouse models and hepatic stellate cells (HSCs), we analyzed the involvement of Thr‐OPN in liver fibrosis. The result showed that, first, Thr‐OPN level was significantly higher in patients with liver cirrhosis than that in patients with chronic hepatitis B and healthy controls. Thr‐OPN level was positively correlated with liver fibrosis degree in clinical samples. Then in mouse models, it showed a similar correlation between hepatic Thr‐OPN levels and liver fibrosis degree. Thr‐OPN peptides exacerbated liver fibrosis in OPN‐deficient mice, whereas the neutralization of Thr‐OPN alleviated liver fibrosis in wild‐type mice. Furthermore, when compared with full‐length OPN (FL‐OPN), Thr‐OPN exhibited a greater ability to promote HSC activation, proliferation, and migration via mitogen‐activated protein (MAP) kinase and nuclear factor (NF)‐κB pathways. In conclusion, Thr‐OPN, not FL‐OPN, was critically involved in the exacerbation of liver fibrosis by α9 and α4 integrins via MAP kinase and NF‐κB signaling pathway, thus representing a novel diagnostic biomarker and treatment target for liver cirrhosis.

Objective. The aim of this study was to investigate the toxigenic Clostridium difficile colonization (CDC, colonization with toxigenic C. difficile but without symptoms) and C. difficile infection (CDI, active C. difficile infection resulting in disease symptoms) in hepatic cirrhosis patients, identify the risk factors of CDC, and determine the correlation between CDC and CDI. Methodology. The strains of toxigenic C. difficile were isolated from patients with hepatic cirrhosis within 48 h after admission, followed by multilocus sequence typing (MLST). Patients were divided into toxigenic CDC group and noncolonized (NC) group according to the colonization. Logistic regression analysis was performed to analyse the risk factors for the CDC. Besides, the CDI incidence was compared between the two groups. Results. Colonization of toxigenic C. difficile was identified in 104 cases (19.8 %). Eighteen sequence types (STs) were identified, among which ST-3, ST-54, ST-35 and ST-37 were the predominant types. Child-Pugh class C(relative risk, RR, 3.025; 95 % CI: 1.410–6.488), decrease of prothrombin time activity (PTA) (RR 2.180; 95 % CI: 1.368–3.476), decrease of platelet (RR 2.746; 95 % CI: 0.931–8.103) and concurrent hepatic encephalopathy (RR 1.740; 95 % CI: 1.012–2.990) were identified as the risk factors for the hepatic cirrhosis patients with CDC. The CDI incidence in the CDC group was also significantly higher than that of the NC group (26.0 % vs 1.7 %, P

Antioxidative and anti-inflammatory effects of trimetallic nitride endohedral fullerenes carboxyl-Gd3N@C80, a newly developed magnetic resonance imaging (MRI) contrast agent, were investigated. All hydrochalarone and carboxyl-functionalized fullerenes showed effective radical (hydroxyl and superoxide anion) scavenging, whereas the carboxyl-Gd3N@C80 more efficiently attenuated lipopolysaccharide (LPS) induced oxidative stress in macrophages. Carboxyl-Gd3N@C80 also suppressed LPS-elicited mRNA expression of pro-inflammatory inducible nitric oxide synthase and tumor necrosis factor-alpha, and upregulated antioxidative enzyme axis Nrf2 and heme oxygenase-1, possibly via ERK but not AKT signaling pathways. Therefore, carboxyl-Gd3N@C80 held a great promise in becoming a novel theranostic nanoplatform for simultaneously deliver MRI contrast and therapeutic functions to inflammation-related diseases.

Differential diagnosis of chronic post-traumatic osteomyelitis (CPO) from aseptic inflammation remains challenging, since both pathological processes share similar clinical symptoms. Here we utilized a novel targeted metallofullerene nanoparticle based magnetic resonance imaging (MRI) probe IL-13-TAMRA-Gd3N@C80(OH)30− (CH2CH2COOH)20 to detect CPO in mouse tibia via overexpressed IL-13Rα2 receptors. The functionalized metallofullerene was characterized by X-ray photoelectron spectroscopy. Upon lipopolysaccharide (LPS) stimulation, macrophage Raw 264.7 cells showed elevated IL-13Rα2 expression via immunofluorescence staining and increased MRI probe binding via built-in TAMRA fluorescence imaging. Trauma was induced in both tibia of mice and bacteria soaked suture was inserted into the right tibia to initiate infection. During the acute phase (1.5 weeks), luminol-bioluminescence imaging revealed much higher myeloperoxidase activity in the infected tibia compared to the sham. In the chronic phase (4 weeks), X-ray radiography illustrated bone deformation in the infected tibia compared to the sham. With T1 weighted sequences, the probe clearly exhibited hyperintensity in the infection foci at both acute and chronic phases, which was not observed in the sham tibia. Histological analysis revealed severe bone structural destruction and massive inflammatory cell infiltration in the infected tibia. Immunohistochemistry confirmed abundant expression of IL-13Rα2 in the infection site. In summary, we developed a noninvasive imaging approach to detect and differentiate CPO from aseptic inflammation using a new IL-13Rα2 targeted metallofullerene MRI probe. In addition, for the first time, IL-13Rα2 was investigated as a unique biomarker in the context of osteomyelitis. Our data established a foundation for the translational application of this MRI probe in the clinical differentiation of CPO.

The adaptive immune system uses several strategies to generate a repertoire of T cell receptors (TCR) with sufficient diversity to recognize the universe of potential pathogens. However, it remains unclear how differences in the T cell receptor (TCR) contribute to heterogeneity in T cell state. In this study, we used polychromatic flow cytometry to isolate highly pure CD4+/CD8+ naive and memory T cells, and applied deep sequencing to characterize corresponding TCR β-chain (TCRβ) complementary-determining region 3 (CDR3) repertoires. We find that shorter TCRβ CDR3s with fewer insertions were highly enriched during thymic selection. Antigen-experienced T cells (memory T cells) harbor shorter CDR3s vs. naive T cells. Moreover, the public TCRβ CDR3 clonotypes within cell subsets or interindividual tend to have shorter CDR3 length and a significantly larger size compared with “private” clonotypes. Taken together, shorter CDR3s highly enriched during thymic selection and antigen-driven selection, and further enriched in public T-cell responses. These results indicated that it may be evolutionary pressures drive short CDR3s to recognize most of antigen in nature.

The relationship between hemostatic system and HBeAg seroconversion (SC) of chronic hepatitis B (CHB) patients is ill-defined. We therefore evaluate the predictive value of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) and VWF (von Willebrand factor) for CHB patients during 5-year entecavir (ETV) treatment. One hundred and fourteen HBeAg positive CHB patients on continuous ETV treatment were recruited. Liver biopsies were evaluated using the METAVIR scoring system, and plasma ADAMTS13 activity (ADAMTS13: AC) and VWF antigen (VWF: Ag) were determined at baseline, 3, 12, 24, 36, and 60 months, respectively. ETV treatment resulted in an increased ADAMTS13: AC and decreased VWF: Ag (both P P

T cell receptor beta variable (TCRBV) repertoire could imply the composition and function status of T cells in subjects with HBV infection. The gene melting spectral pattern (GMSP) can be used to determine the profile of TCRBV gene family. The molecular profile of TCRBV in peripheral lymphocytes from asymptomatic HBV carriers (AsC) remains ill-defined. Peripheral blood mononuclear cells (PBMCs) were separated and sorted, and the profiles of TCRBV complementarity-determining region 3 (CDR3) in CD4(+), CD8(+) T subsets and PBMCs were assayed using GMSP. The number of skewed TCRBV in the PBMCs was significantly lower than that in the CD4(+) or CD8(+) T subsets, and the number of monoclonal TCRBV families in the CD8(+) T subset was significantly higher than that in CD4(+) T subset. Compared to healthy donors, TCRBV11, BV13.1, BV20 and BV24 were used more frequently than other TCRBV members in PBMCs from AsC subjects. Furthermore, the relatively conserved CDR3 motifs were detected in these TCRBVs. The results indicate that the T cell response in AsC subjects involves several TCRBVs, and that the CD8(+) T subset maybe more relevant to pathogenesis of AsC. Moreover, the four relative conserved TCRBVs maybe a target for personalized treatments for persistent HBV infection.

Circulating immune complexes (CICs) are produced during the immune response. It is more clinically important to establish a general and efficient CICs dissociation technique for the detection of antigens for CICs other than the detection of free antigens in the serum. Polyethylene glycol (PEG) two-precipitation separation and glycine-HCl as a buffer system were employed to develop a general and efficient buffer dissociation technique to separate CICs from serum and dissociate antigens from CICs. The measurement value of new PEG two-precipitation separation technique was higher than traditional PEG precipitation separation technique. There were slight differences in the dissociation conditions of HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC as compared to HBsAg-IC. The detection of antigens in HBsAg-IC, HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC with this technique was superior to that with HCl Dissociation, Trypsin Digestion or Immune Complex Transfer technique. PEG two-precipitation dissociation technique may reduce macromolecular protein and the adhesion of free antigens during the co-precipitation, which increases the efficiency of separation and precipitation of CICs. This technique also avoids the damage of reagents to antigens, assuring the repeatability, reliability and validity. Thus, this technique is application in samples negative or positive for free antigens.

Antioxidative and anti-inflammatory effects of trimetallic nitride endohedral fullerenes carboxyl-Gd3N@C80, a newly developed magnetic resonance imaging (MRI) contrast agent, were investigated. All hydrochalarone and carboxyl-functionalized fullerenes showed effective radical (hydroxyl and superoxide anion) scavenging, whereas the carboxyl-Gd3N@C80 more efficiently attenuated lipopolysaccharide (LPS) induced oxidative stress in macrophages. Carboxyl-Gd3N@C80 also suppressed LPS-elicited mRNA expression of pro-inflammatory inducible nitric oxide synthase and tumor necrosis factor-alpha, and upregulated antioxidative enzyme axis Nrf2 and heme oxygenase-1, possibly via ERK but not AKT signaling pathways. Therefore, carboxyl-Gd3N@C80 held a great promise in becoming a novel theranostic nanoplatform for simultaneously deliver MRI contrast and therapeutic functions to inflammation-related diseases.

Supplemental Digital Content is available in the text, Effective antiviral therapy plays a key role in slowing the progression of chronic hepatitis B (CHB). Identification of serum indices, including hepatitis B e antigen (HBeAg) expression and seroconversion, will facilitate evaluation of the efficacy of antiviral therapy in HBeAg-positive CHB patients. The biochemical, serological, virological parameters, and the frequency of circulating CD4+CD25+ regulatory T cell (Treg) in 32 patients were measured at baseline and every 12 weeks during 96 weeks of tenofovir disoproxil fumarate (TDF) treatment. The relationship between the hepatitis B virus (HBV) deoxyribonucleic acid (DNA) and Treg and alanine aminotransferase (ALT) levels was analyzed, respectively. The molecular profiles of T-cell receptor beta variable chain (TRBV) were determined using gene melting spectral pattern. For the seroconverted 12 patients, ALT declined to normal levels by week 24 and remained at this level in subsequent treatment; moreover, the predictive cutoff value of ALT for HBeAg seroconversion (SC) was 41.5 U/L at week 24. The positive correlation between HBV DNA and Treg and ALT was significant in SC patients, but not in non-SC patients. Six TRBV families (BV3, BV11, BV12, BV14, BV20, and BV24) were predominantly expressed in SC patients at baseline. The decline of ALT could be used to predict HBeAg seroconversion for CHB patients during TDF treatment. In addition, the profile of Tregs and TRBVs may be associated with HBeAg seroconversion and could also be a potential indicator for predicting HBeAg SC and treatment outcome for CHB patients.

Immunization with recombinant HBV vaccine induces specific immune responses in human causing B lymphocytes to produce protective HBsAb, and to form memory B lymphocytes, thereby facilitating HBV immunity in the body. However, B lymphocytes heterogeneity and characteristics are not fully elucidated. In this study, we conducted high-throughput sequencing of BCR heavy chain CDR3 repertoires in 3 healthy volunteers before and after the third immunization with recombinant HBV vaccine. We used Roche 454 Genome Sequencer FLX system to perform a comparative analysis of IgM and IgG H chain CDR3 repertoires. First, we found that the diversity of IgG H chain CDR3 repertoires was 1/6 of IgM on average. Moreover, after the third immunization with HBV vaccine, the diversity of IgG H chain CDR3 repertoires was 1/26 of IgM on average. Second, we detected relatively high levels of HBsAbs in all the healthy volunteers after immunization with HBV vaccine. The volunteers shared a small number of CDR3 sequences before and after immunization, and among each other. However, we did not find completely identical BCR H chain CDR3 amino acid sequences in these volunteers. Lastly, after immunization with recombinant HBV vaccine, the volunteers showed high frequency of IgG H chain CDR3 amino acid sequences mostly resulting from rearrangements of IGHV, IGHD and IGHJ, suggesting that the mechanism of high frequency CDR3 generation might be associated with the maturation of IgG affinity (somatic hypermutation) during the recombinant HBV vaccine-induced B lymphocyte responses. This study identified the characteristics and changes of BCR CDR3 repertoires before and after immunization with HBV vaccine, and evaluated the performance of the sequencing technology for this application. Our findings provide a basis for further research in B lymphocyte generated HBsAb heterogeneity and monitoring the maintenance of memory B lymphocytes.

The profile of T-cell receptor beta-chain variable (TRBV) genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern (GMSP) can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection (AHI), peripheral blood mononuclear cells (PBMCs) were separated and further sorted into CD4+ and CD8+ T-cell subsets. The molecular features of the TRBV complementary determining region 3 (CDR3) motifs were determined using GMSP analysis. When a GMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4+ and CD8+ T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8+ T-cell subset was significantly higher than that of the CD4+ T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4+ and CD8+ T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8+ T-cell subset, may be relevant to the pathogenesis of subjects with AHI. The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.

Background: The microbiota plays a critical role in the process of human carcinogenesis. Pancreatic head carcinoma (PHC)-associated tongue coating microbiome dysbiosis has not yet been clearly defined.Objective: Our aim is to reveal the bacterial composition shifts in the microbiota of the tongue coat of PHC patients.Design: The tongue coating microbiota was analyzed in 30 PHC patients and 25 healthy controls using 16S rRNA gene sequencing technology.Results: The microbiome diversity of the tongue coat in PHC patients was significantly increased, as shown by the Shannon, Simpson, inverse Simpson, Obs and incidence-based coverage estimators. Principal component analysis revealed that PHC patients were colonized by remarkably different tongue coating microbiota than healthy controls and liver cancer patients. Linear discriminant analysis effect size revealed that Leptotrichia, Fusobacterium,Rothia, Actinomyces, Corynebacterium, Atopobium, Peptostreptococcus, Catonella, Oribacterium, Filifactor, Campylobacter, Moraxella and Tannerella were overrepresented in the tongue coating of PHC patients, and Haemophilus, Porphyromonas and Paraprevotella were enriched in the tongue coating microbiota of healthy controls. Strikingly, Haemophilus, Porphyromonas, Leptotrichia and Fusobacterium could distinguish PHC patients from healthy subjects, and Streptococcus and SR1 could distinguish PHC patients from liver cancer patients. Conclusions: These findings identified the microbiota dysbiosis of the tongue coat in PHC patients, and provide insight into the association between the human microbiome and pancreatic cancer.

Understanding the composition of the microbial populations in the intestines of liver transplant patients is important to preventing postoperative infection. We investigated the relationship between the risk of postoperative infection and variation in the predominant fecal microbial composition during the perioperative period. We prospectively analyzed the predominant intestinal microbiome of five asymptomatic adult carriers of hepatitis B virus (as controls without any antibiotics) at four weekly follow-up visits and 12 patients before operation and at three weekly postoperative follow-up visits within the first month. Analysis was by denaturing gradient gel electrophoresis (DGGE) and sequencing with digital processing of DGGE profiles using BioNumerics software. Our results showed that the predominant intestinal microbial diversity decreased substantially in eight patients during the perioperative period. Among these, five patients experienced infection with a postoperative hospital stay of more than 30 days. The rest of the four patients who experienced shorter postoperative hospital stays showed only slight variation in predominant intestinal bacterial composition and temporal stability similar to asymptomatic controls. Postoperative fecal DGGE profiles showed mostly bands assigned to Bacteroides and Firmicutes. We conclude that an empiric prophylaxis strategy that destructs gut microecological balance will not be effective in reducing the risk of postoperative infection. Instead, the destruction of intestinal microbiota might result in the appearance of opportunistic pathogens such as Bifidobacterium dentium which rarely appears in the intestinal DGGE profiles of normal humans. Cognizance of the variation of intestinal microbial profiles during the perioperative period is a critical aspect of caring for liver transplant recipients.

The aim of this study was to determine the correlation between dynamic changes in serum cytokine/chemokine expression levels in response to entecavir (ETV) treatment and HBV e antigen (HBeAg) seroconversion in patients with chronic hepatitis B (CHB). Four cytokines (interleukin [IL]-4, IL-6, IL-8, and interferon-γ) and five chemokines (macro-phage inflammatory protein [MIP]-1α, MIP-1β, platelet derived growth factor-BB, and interferon-inducible protein 10 [IP-10]) before ETV therapy and at 3, 6, 12, 24, 36 and 60 months during therapy in 105 CHB patients were analyzed. The results showed that the low decrease rate of IP-10 levels after 1 year of ETV treatment was an independent predictor of HBeAg seroconversion at year 5 (Hazard ratio = 0.972). The area under the receiver operating characteristic curves for the decrease rate of IP-10 levels after 1 year of treatment to discriminate a year-5 HBeAg seroconversion was 0.752 (p = 0.005). The results indicate that higher IP-10 level at year one of ETV treatment is associated with an increased probability of HBeAg seroconversion. Quantification of IP-10 during ETV treatment may help to predict long-term HBeAg seroconversion in patients with CHB.

Glycolipids are potent activator of natural killer T (NKT) cells. The relationship between NKT cells and intestinal bacterial glycolipids in liver disorders remained unclear. We found that, in sharp contrast to specific pathogen-free (SPF) mice, germ-free (GF) mice are resistant to Concanavalin A (ConA)-induced liver injury. ConA treatment failed to trigger the activation of hepatic NKT cells in GF mice. These defects correlated with the sharply reduced levels of CD1d-presented glycolipid antigens in ConA-treated GF mice compared with SPF counterparts. Nevertheless, CD1d expression was similar between these two kinds of mice. The absence of intestinal bacteria did not affect the incidence of αGalCer-induced liver injury in GF mice. Importantly, we found the intestinal bacteria contain glycolipids which can be presented by CD1d and recognized by NKT cells. Furthermore, supplement of killed intestinal bacteria was able to restore ConA-mediated NKT cell activation and liver injury in GF mice. Our results suggest that glycolipid antigens derived from intestinal commensal bacteria are important hepatic NKT cell agonist and these antigens are required for the activation of NKT cells during ConA-induced liver injury. These finding provide a mechanistic explanation for the capacity of intestinal microflora to control liver inflammation.

Scientific Reports 5: Article number: 10942; published online: 01 June 2015; updated: 23 February 2016 This Article contains typographical errors in Table 2 where ‘Week 2 (N = 32)’ was incorrectly given as ‘Week (N = 2)’.

This study aimed to determine the degree of clonal expansion of T cells in peripheral blood mononuclear cells (PBMCs) isolated from patients suffering from different clinical types of hepatitis B (HB) infection and to analyse the clinical relevance of the skewed T-cell receptor beta variable (TCRBV). Sera and PBMCs were collected from 90 HB patients. Gene melting spectral pattern (GMSP) analysis was used to determine the distribution and expansion of populations expressing specific TCRBV complementary determined region 3 (CDR3) genes. TCRBV genes associated with monoclonal expansion were sequenced. TCRBV families from the majority of patients (80/90) displayed skewed T-cell expansion. Furthermore, TCRBV11, BV12 and BV13.1 were more frequent than other TCRBV genes; the sequence of TCRBV11 CDR3 was expressed as 'VYNEQ' in all patients and was accompanied by the BJ2.1 fragment. In patients with chronic HB, the frequency of skewed TCRBV was inversely correlated with hepatitis B virus (HBV) DNA levels. The persistently skewed TCRBV gene families in HB patients may be associated with the development and maintenance of hepatitis. GMSP analysis of TCRBV gene families may be helpful in estimating disease status, and BV11 may be associated with HBV replication in patients with chronic HBV infection.

CD4 + CD25 + regulatory T-cells: a potential target for treating patients with chronic HBV infection

Secondary bacterial lung infection (SBLI) is a serious complication in patients with H7N9 virus infection, and increases disease severity. The oropharyngeal (OP) microbiome helps prevent colonisation of respiratory pathogens. We aimed to investigate the OP microbiome of H7N9 patients with/without secondary bacterial pneumonia using 16S rRNA gene sequencing. OP swab samples were collected from 51 H7N9 patients (21 with SBLI and 30 without) and 30 matched healthy controls (HCs) and used for comparative composition, diversity and richness analyses of microbial communities. Principal coordinates analysis successfully distinguished between the OP microbiomes of H7N9 patients and healthy subjects, and the OP microbiome diversity of patients with SBLI was significantly increased. There was significant dysbiosis of the OP microbiome in H7N9 patients, with an abundance of Leptotrichia, Oribacterium, Streptococcus, Atopobium, Eubacterium, Solobacterium and Rothia species in patients with SBLI, and Filifactor, Megasphaera and Leptotrichia species in patients without SBLI, when compared with HCs. Importantly, Haemophilus and Bacteroides species were enriched in HCs. These findings revealed dysbiosis of the OP microbiota in H7N9 patients, and identified OP microbial risk indicators of SBLI, suggesting that the OP microbiome could provide novel and non-invasive diagnostic biomarkers for early microbiota-targeted prophylactic therapies for SBLI prevention.

Coagulative and fibrinolytic disorders appear to be associated with the development of lung cancer. The aim of the present study was to determine plasma levels of von Willebrand factor (VWF) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 13 (ADAMTS-13), and factor VIII (FVIII) activity, in association with O and non-O blood groups in patients with lung cancer. Plasma levels of VWF and ADAMTS-13, and FVIII activity were measured in 115 patients with lung cancer and 98 healthy subjects. Phenotyping of the ABO blood groups was also performed for the two groups. Significantly increased VWF levels and FVIII activity, as well as significantly decreased ADAMTS-13 levels, were observed in patients with distant metastasis as compared with those without distant metastasis and the healthy controls. Plasma VWF levels and FVIII activity were significantly increased in subjects with non-O type blood compared with those with type O blood in the two groups. However, a significant decrease in ADAMTS-13 levels was observed only in the control group among those with non-O type blood, compared with those with type O blood. The results of the present study indicate that increased VWF and decreased ADAMTS-13 levels facilitate the invasiveness and metastasis of lung cancer. Non-O blood groups constitute a risk factor for increased VWF and FVIII in plasma. Continued monitoring of VWF and ADAMTS-13 levels, and of FVIII activity in patients with lung cancer with distinct blood groups may help to minimize the incidence of thrombotic events and improve assessment of disease progression.

The novel avian origin influenza A (H7N9) virus has caused severe diseases in humans in eastern China since the spring of 2013. Fatal outcomes of H7N9 infections are often attributed to the severe pneumonia and acute respiratory distress syndrome (ARDS). There is urgent need to discover biomarkers predicting the progression of disease and fatal outcome of potentially lethal flu infections, based on sound statistical analysis. We discovered that 34 of the 48 cytokines and chemokines examined in this study were significantly elevated in the plasma samples from patients infected with H7N9. We report for the first time that the levels of MIF, SCF, MCP-1, HGF and SCGF-β are highly positively linked to disease severity and the profile of mediators MIF, SCF, MCP-1, HGF, SCGF-β, IP-10, IL-18 and IFN-γ is an independent outcome predictor.

Background Several studies on the association of TNF-alpha (−308 G/A), IL-6 (−174 G/C) and IL-1beta (−511 C/T) polymorphisms with polycystic ovary syndrome (PCOS) risk have reported conflicting results. The aim of the present study was to assess these associations by meta-analysis. Results A total of 14 eligible articles (1665 cases/1687 controls) were included in this meta-analysis. The results suggested that there was no obvious association between the TNF-alpha (−308 G/A) polymorphism and PCOS in the overall population or subgroup analysis by ethnicity, Hardy–Weinberg equilibrium (HWE) in controls, genotyping method, PCOS diagnosis criteria, and study sample size. Also, no obvious association was found between the TNF-alpha (−308 G/A) polymorphism and obesity in patients with PCOS (body mass index [BMI] ≥ 25 kg/m2 vs. BMI

A novel influenza A (H7N9) virus of avian origin emerged in eastern China in the spring of 2013. This virus causes severe disease in humans, including acute and often lethal respiratory failure. As of January 2014, 275 cases of H7N9-infected patients had been reported, highlighting the urgency of identifying biomarkers for predicting disease severity and fatal outcomes. Here, we show that plasma levels of angiotensin II, a major regulatory peptide of the renin–angiotensin system, are markedly elevated in H7N9 patients and are associated with disease progression. Moreover, the sustained high levels of angiotensin II in these patients are strongly correlated with mortality. The predictive value of angiotensin II is higher than that of C-reactive protein and some clinical parameters such as the PaO2/FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen). Our findings indicate that angiotensin II is a biomarker for lethality in flu infections. Supplementary information The online version of this article (doi:10.1038/ncomms4595) contains supplementary material, which is available to authorized users., An avian influenza H7N9 virus causes severe human disease, including acute and often lethal respiratory failure. Here, the authors report that plasma levels of angiotensin II, a regulatory peptide of the renin–angiotensin system, are associated with disease severity and fatal outcome in infected patients. Supplementary information The online version of this article (doi:10.1038/ncomms4595) contains supplementary material, which is available to authorized users.

Some dielectric oxides have been synthesized and characterized in the BaO–Nd 2 O 3 –TiO 2 –Ta 2 O 5 system. Through X-ray powder diffraction analysis, Ba 4 Nd 2 Ti 4 Ta 6 O 30 and Ba 5 NdTi 3 Ta 7 O 30 are identified as tetragonal tungsten–bronze compounds, and their lattice constants are a = b =12.4007(7) A, c =3.9030(3) A and a = b =12.4826(6) A, c =3.9295(5) A, respectively. The possible space group is P 4 bm . The polycrystalline samples of these compounds exhibit high dielectric constant (137 ∼ 160) and low dielectric loss, tan δ −4 at 1 MHz.