Profiling of the pattern of the human TRB/IGH-CDR3 repertoire in primary biliary cholangitis patients.

INTRODUCTION: Primary biliary cholangitis (PBC) is characterized by lymphocyte cell-induced immune destruction of cholangiole. However, the immunological characteristics of peripheral blood cells in PBC patients remain unknown. This study was designed to reveal the differences in the immunological characteristics between PBC patients and healthy adults. METHODS: We performed high-throughput sequencing to determine the TRB-CDR3 and IGH-CDR3 repertoires of T and B cells in 19 healthy controls and 29 PBC patients. Different immunological characteristics, such as distinctive complementarity determining region 3 (TRB-CDR3) lengths, usage bias of V and J segments, and random nucleotide addition were identified in PBC and healthy control (HC) groups. RESULTS: The diversity of TRB-CDR3 was significantly lower in the PBC group compared with the HC group. CDR3 and the N addition length distribution were significantly changed compared with the HC group. It appeared that the PBC group had more short N additions and the HC group had more long N additions in the TRB-CDR3 repertoire. The results also revealed a set of PBC-associated clonotypes compared with the HC group. CONCLUSION: This study suggested that PBC is a complex autoimmune disease process with evidence of different TRB-CDR3 rearrangements compared with healthy adults that share IGH-CDR3 peptides with some autoimmune diseases. This new insight may contribute to a better understanding of the immune functions of PBC patients and benefit efficient applications of PBC diagnosis and treatments.

Increased plasma olfactomedin 2 after interventional therapy is a predictor for restenosis in lower extremity arteriosclerosis obliterans patients.

Animal studies have indicated that olfactomedin 2 (OLFM2) is involved in the process of vascular remolding. The aim of the present study was to investigate circulating OLFM2 levels in lower extremity arteriosclerosis obliterans (LEASO) patients and the association of OLFM2 with postoperative restenosis in patients. A total of 203 LEASO patients were enrolled in the present study. Plasma OLFM2 was measured before and 6 h after interventional therapy. After 6 months, patients were divided into a restenosis group and a non-restenosis group. Inter-group and intra-group differences in plasma OLFM2 were compared. The correlation between plasma OLFM2 and the severity of restenosis was analyzed by Spearman's correlation analysis. An receiver operating characteristic (ROC) curve was used to evaluate the predictive efficacy of plasma OLFM2 on restenosis. Logistic regression was used to determine the risk factors for restenosis. Postoperative OLFM2 in the restenosis group was significantly higher compared with the non-restenosis group (34.07 ± 5.76 ng/mL vs. 19.53 ± 2.99 ng/mL). No significant difference in preoperative plasma OLFM2 levels was identified between the two groups (10.92 ± 2.49 ng/mL vs. 11.54 ± 3.18 ng/mL). Postoperative OLFM2 levels were positively correlated with the severity of restenosis (r = 0.728, p < .001). The area under the ROC curve was 0.902 (95% confidence interval (CI): 0.874-0.965), with a cutoff value of 26.91 ng/mL (95% CI: 26.16-28.32). Plasma OLFM2 was an independent risk factor for restenosis. Our results suggest that plasma OLFM2 is a potential biomarker for restenosis and may be a novel target for the treatment of restenosis.

Functional Variants Identified Efficiently through an Integrated Transcriptome and Epigenome Analysis.

Although genome-wide association studies (GWAS) have identified numerous genetic loci associated with complex diseases, the underlying molecular mechanisms of how these loci contribute to disease pathogenesis remain largely unknown, due to the lack of an efficient strategy to identify these risk variants. Here, we proposed a new strategy termed integrated transcriptome and epigenome analysis (iTEA) to identify functional genetic variants in non-coding elements. We considered type 2 diabetes mellitus as a model and identified a well-known diabetic risk variant rs35767 using iTEA. Furthermore, we discovered a new functional SNP, rs815815, involved in glucose metabolism. Our study provides an approach to directly and quickly identify functional genetic variants in type 2 diabetes mellitus, and this approach can be extended to study other complex diseases.

The efficacy of tranexamic acid in reducing blood loss in total shoulder arthroplasty: A meta-analysis.

BACKGROUND: The purpose of this meta-analysis is to compare the efficacy of tranexamic acid (TXA) versus placebo after a total shoulder arthroplasty (TSA). METHODS: In April 2017, a systematic computer-based search was conducted in the databases of PubMed, Embase, Web of Science, Cochrane Library, and Google. Studies comparing TXA versus placebo in reducing blood loss after TSA were included. The endpoints were the need for transfusion, blood loss in drainage, hemoglobin drop, and total blood loss. Stata 12.0 software was used for the meta-analysis. RESULTS: Six studies involving a total of 637 patients met the inclusion criteria. The meta-analysis revealed that, compared with control groups, treatment with TXA could decrease the need for transfusion (P < .00001), blood loss in drainage (P = .000), hemoglobin drop (P = .001), and total blood loss (P = .000). CONCLUSION: TXA can decrease the need for transfusion as well as total blood loss in TSA patients. There was a negative correlation between the TXA dose and the need for transfusion and blood loss in drainage. Because the administration route and the dose of TXA were different, more studies are needed in order to identify the optimal dose and route.

Wnt signaling promotes hindgut fate commitment through regulating multi-lineage genes during hESC differentiation.

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here, we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes, respectively. Clusters of Wnt targets showed enrichment in specific biological functions, such as "gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover, we adopted a high-throughput chromatin immunoprecipitation and deep sequencing (ChIP-seq) approach to identify the genomic regions through which Wnt-activated transcription factor TCF7L2 regulated transcription. We identified 83 Wnt direct target candidates, including the hindgut marker CDX2 and the genes relevant to morphogenesis (MSX1, MSX2, LEF1, T, PDGFRB etc.) through combinatorial analysis of the RNA-seq and ChIP-seq data. Together, our study identified a series of direct and indirect Wnt targets in hindgut differentiation, and uncovered the diverse mechanisms of Wnt signaling in regulating multi-lineage differentiation.

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2.

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD(+)-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.

PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs), but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs, binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7, which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database, we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore, we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together, we uncover a potential mechanism of how PTEN safeguards NSCs, and establish a cellular platform to identify factors involved in NSC transformation, potentially permitting personalized treatment of glioblastoma.

Progress and prospects in stem cell therapy.

In the past few years, progress being made in stem cell studies has incontestably led to the hope of developing cell replacement based therapy for diseases deficient in effective treatment by conventional ways. The induced pluripotent stem cells (iPSCs) are of great interest of cell therapy research because of their unrestricted self-renewal and differentiation potentials. Proof of principle studies have successfully demonstrated that iPSCs technology would substantially benefit clinical studies in various areas, including neurological disorders, hematologic diseases, cardiac diseases, liver diseases and etc. On top of this, latest advances of gene editing technologies have vigorously endorsed the possibility of obtaining disease-free autologous cells from patient specific iPSCs. Here in this review, we summarize current progress of stem cell therapy research with special enthusiasm in iPSCs studies. In addition, we compare current gene editing technologies and discuss their potential implications in clinic application in the future.

Oncogenic function of DACT1 in colon cancer through the regulation of ß-catenin.

The Wnt/ß-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/ß-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of ß-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of ß-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated ß-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3ß and ß-catenin. DACT1 stabilizes ß-catenin via DACT1-induced effects on GSK-3ß and directly interacts with ß-catenin proteins. The level of phosphorylated GSK-3ß at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of ß-catenin via increasing the level of phosphorylated GSK-3ß at Ser9 to inhibit the activity of GSK-3ß. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the ß-catenin-dependent pathway.

[Synergic effects of Danggui Buxue Decoction in reducing toxicity of cytoxan in tumor-bearing mice].

OBJECTIVE: To study the inhibitory effect of Danggui Buxue Tang (DBT), a compound traditional Chinese herbal medicine for supplementing blood, on tumor growth in tumor-bearing mice after inoculation of EL-4 cells, and its immune mechanism as well as its synergic effect in reducing toxicity of cytoxan (CTX). METHODS: Experiment was carried out in tumor-bearing mice after inoculation of EL-4 cells. The mice were randomly divided into four groups after 7 days of the inoculation: untreated group, DBT-treated group [24 g/(kg x d)], CTX-treated group [7.5 mg/(kg x d)] and DBT plus CTX-treated group, with another ten normal mice as control. Inhibitory rate of tumor growth, survival time, immune function and variability of blood cells were measured in the mice during the experiment. RESULTS: After treatment of relevant interventions for 15 days, the tumor in the DBT-treated group, CTX-treated group and DBT plus CTX-treated group grew slower than the untreated group (P<0.05). Murine survival time in the DBT-treated group, CTX-treated group and DBT plus CTX-treated group was lengthened as compared with that in the untreated group (P<0.05). Compared with the untreated group, all kinds of immune indexes in the DBT-treated group and DBT plus CTX-treated group were significantly improved (P<0.05), while the immune indexes in the CTX-treated group were decreased (P<0.05). Compared with the CTX-treated group, all kinds of immune indexes in the DBT plus CTX-treated group were significantly improved (P<0.05). CONCLUSIONS: DBT can enhance the immune function in tumor-bearing mice and the inhibitory effect of DBT on tumor growth is related to the enhanced immune response. DBT can also increase the therapeutic effects and reduce the side effects of CTX.

[Induction of skin allograft tolerance in mice by using anti-alphabeta T cell receptor and anti-CD80 monoclonal antibodies combined with bone marrow transfusion].

OBJECTIVE: To explore the role of anti-alphabeta T cell receptor (TCR) and anti-CD80 monoclonal antibodies (mAbs) combined with donor bone marrow cells (BMCs) infusion in the induction of murine skin allografts tolerance. METHODS: On day 0, 2 x 10(8) BMCs of BALB/c mice were injected into recipient C57BL/6 mice via the tail vein, meanwhile, an intraperitoneal injection of TCRalphabeta mAb (500 microg) was given. On day 2, CD80 mAb was administered intraperitoneally. Skin grafting was performed on day 6. Delayed type hypersensitivity (DTH), mixed lymphocyte reaction (MLR), IL-2 reverse assay of MLR, adoptive transfer assay and chimerism detection were performed at different time points and tolerance mechanisms were investigated. RESULTS: The mean survival time (MST) of BALB/c skin allografts in C57BL/6 recipients that were treated by anti-TCRalphabeta and anti-CD80 mAbs combined with donor BMCs infusion was 70 days. DTH and MLR assay indicated that the tolerant mice displayed significant hyporesponsiveness. The result of IL-2 reverse test showed that clone anergy was probably involved in the formation of tolerance in the tolerant C57BL/6 mice. In vivo and in vitro adoptive transfer assay, suppressive activity in the spleens of tolerant C57BL/6 mice was observed. Chimerism existed in both the thymus and spleen of the tolerant C57BL/6 mice. The chimerism level gradually declined with time. CONCLUSION: Treatment of anti-TCRalphabeta and anti-CD80 mAbs combined with donor BMCs infusion can successfully induce a long-term tolerance in BALB/c mice to C57BL/6 skin graft. Multiple mechanisms, including clone anergy, suppressor cells and chimerism are involved in the tolerance.

[Characterization of a novel transplantable orthotopic nude mouse model with xenografted human bladder transitional cell tumor (BIU-87)].

OBJECTIVE: A mouse model of orthotopic bladder cancer simulating its human counterpart is of great importance in preclinical evaluation of new treatment modalities such as immunotxin therapy. The aim of the present study is to establish a novel nude mouse model with xenografted human bladder cancer. METHODS: Single cell suspension of an established human bladder transitional cell carcinoma (TCC) cell line BIU-87 was instilled into nude mouse bladders which were pretreated with mild acid washing. The tumor growth in mouse bladder was assessed weekly by magnetic resonance imaging (MRI). At intervals following implantation and MRI tumor detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies. RESULTS: The overall tumor establishment was 92.9% (52/56 mice) at 7 - 36 days, while in the subgroup of animals sacrificed at 12 - 13 days, 40 out of 42 animals (95.2%) developed TCC, the majority of which was superficial. The tumor stages were assessed by gross and histopathology. Histological examination confirmed the presence of grade II - III TCC. Immunocytochemistry confirmed that the tumor model maintained the biological and immunological features of BIU-87 cells. The changes seen on MRI images well correlated with the extent of tumor invasion identified by histology. Carcinoma in situ could be detected histologically at 7 - 9 days post-inoculation and progressed into papillary or invasive tumors thereafter. CONCLUSION: The orthotopic BIU-87 TCC model in nude mice is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.

[Adenovirus-mediated CTLA4Ig and OX40Ig gene transfer induces long-term survival of cardiac allografts in rats].

OBJECTIVE: To investigate the potential role of CTLA4Ig gene and OX40Ig protein in inducing transplantation tolerance and the mechanisms thereof. METHODS: Thirty Lewis rats underwent transplantation of the hearts of DA rats and then randomly divided into five equal groups: control group, blank virus AdEGFP treated group (adenovirus containing EGFP at the dose of 1-5 x 10(9) pfu/ml was infused via portal vein immediately after the operation), AdCTLA4Ig treated group, AdOX40Ig treated group, and AdCTLA4Ig-IRES-OX40Ig treated group. The cardiac allograft survival was monitored by daily palpation. The total cessation of beating was defined as rejection and was confirmed by histology. Peripheral venous blood samples were collected 0, 3, 7, 10, 14, 21 and 28 days after the administration of adenovirus. ELISA was used to detect the expression of CTLA4Ig and OX40Ig. Twenty days after the heart transplantation single splenocyte suspension was prepared from surviving Lewis rats to be used as responder. The spleens of the normal donor-DA rats and the third strain DA rats to prepare single cell suspension of the same density to perform mixed lymphocyte reaction (MLR). Then recombinant IL-2 was added into the mixed MLR system to observe t\if the MLR could be reversed. Twenty days after the heart transplantation the splenocytes of the tolerating Lewis rats were injected into the lingual vein of the normal Lewis rats to observe the delayed type hypersensitivity (DTH) of the transferred Lewis rat to normal rat splenocytes. RT-PCR was used to detect the mRNA expression of IL-2, interferon-gamma, IL-4, and IL-10. RESULTS: The survival time of the AdCTLA4Ig-IRES-OX40Ig treated group was 151.5 d +/- 42.6 d, significantly longer than those of the AdOX40Ig treated group (60.2 d +/- 11.4 d (P = 0.003), AdCTLA4Ig (43.2 d +/- 11.1 d, P = 0.0026), control group (5.7 d +/- 0.5 d, P = 0.000 43), and AdEGFP treated group (5.2 d +/- 0.4 d, P = 0.000 43). CTLA4Ig and/or OX40Ig proteins were expressed at a high level in the adenoviral treated rats. Compared with the control group the splenocytes of the AdCTLA4Ig-IRES-OX40Ig, AdCTLA4Ig, and AdOX40Ig treated groups displayed donor-specific hyporesponsiveness (P = 0.0016, 0.0026 and 0.001), which could be partly reversed by the addition of exogenous IL-2. Moreover, the hyporesponsiveness could be transferred to the same strain rats through adoptive transfer. In comparison with the normal controls, the expression of Th1 type cytokines, such as IL-2 and IFN-gamma, was significantly decreased in the tolerating rats and significantly increased in the rats with rejection; however the expression of the Th2 type cytokines, such as IL03 and IL-10, was significantly increased in the tolerating rats and significantly decreased in the rats with rejection, showing a deviation of Th1/Th2 type cytokines. CONCLUSION: AdCTLA4Ig-IRES-OX40Ig-mediated genes transfer renders prolonged expression of CTLA4Ig and OX40Ig in Lewis recipient rats, leading to a long-term survival of cardiac allografts. The induced tolerance is donor-specific, and the mechanisms may be associated with T cell anergy, deviation of Th1/Th2, and the regulatory T cells.