Super-Enhancer Driven LIF/LIFR-STAT3-SOX2 Regulatory Feedback Loop Promotes Cancer Stemness in Head and Neck Squamous Cell Carcinoma.

Super-enhancers (SEs) have been recognized as key epigenetic regulators underlying cancer stemness and malignant traits by aberrant transcriptional control and promising therapeutic targets against human cancers. However, the SE landscape and their roles during head and neck squamous cell carcinoma (HNSCC) development especially in cancer stem cells (CSCs) maintenance remain underexplored yet. Here, we identify leukemia inhibitory factor (LIF)-SE as a representative oncogenic SE to activate LIF transcription in HNSCC. LIF secreted from cancer cells and cancer-associated fibroblasts promotes cancer stemness by driving SOX2 transcription in an autocrine/paracrine manner, respectively. Mechanistically, enhancer elements E1, 2, 4 within LIF-SE recruit SOX2/SMAD3/BRD4/EP300 to facilitate LIF transcription; LIF activates downstream LIFR-STAT3 signaling to drive SOX2 transcription, thus forming a previously unknown regulatory feedback loop (LIF-SE-LIF/LIFR-STAT3-SOX2) to maintain LIF overexpression and CSCs stemness. Clinically, increased LIF abundance in clinical samples correlate with malignant clinicopathological features and patient prognosis; higher LIF concentrations in presurgical plasma dramatically diminish following cancer eradication. Therapeutically, pharmacological targeting LIF-SE-LIF/LIFR-STAT3 significantly impairs tumor growth and reduces CSC subpopulations in xenograft and PDX models. Our findings reveal a hitherto uncharacterized LIF-SE-mediated auto-regulatory loop in regulating HNSCC stemness and highlight LIF as a novel noninvasive biomarker and potential therapeutic target for HNSCC.

[Triplet anti-tumor therapy based on thymosin α-1 attenuates incidence of hepatoma and serum alpha-fetoprotein level in rat hepatoma model].

OBJECTIVE: To explore the impact of triple anti-tumor therapy based on thymosin α1 (Tα1) combined with Huaier granule(HG) and sirolimus on the level of serum alpha-fetoprotein (AFP) in rat models of liver cancer. METHODS: Ninety Sprague-Dawley rats were randomly divided into triple anti-tumor therapy group, Tα1 group, HG group, sirolimus group, positive control and blank control groups, with 15 rats in each group. Except the blank control group, the rats in the other groups were induced using diethylnitrosamine (DEN) to establish liver cancer models. After DEN treatment, the triple therapy group underwent 0.8 mg/kg Tα1 subcutaneous injection (from once a day for two weeks to twice a week since the third week), 0.35 g/kg HG gavage (three times a day) and 1 mg/kg sirolimus gavage (once a day). The dose of the rest single drug groups were the same with that of the triple therapy group. The positive control and blank control groups were not treated with the drugs. The treatment lasted 20 weeks. Then, the behavior of the rats were observed at the different time points, and the level of serum AFP in the rats were detected at 6, 16, 18, 20 weeks, respectively. RESULTS: The typical symptoms of liver cancer were seen in the DEN-induced rats at 16 weeks. Since the tenth week, 6 rats died one after another. Pathological section of rat liver tissue suggested that the rat models were established successfully. According to the incidence rate of liver cancer and the survival rate at 20 weeks, the triple anti-tumor therapy was significantly superior to the single drug treatments. In addition, the triple anti-tumor therapy significantly reduced the level of serum AFP in the rats. CONCLUSION: The triple anti-tumor therapy can significantly prolong the survival time of rats with liver cancer, decrease the cancer incidence rate and the level of serum AFP.

[Expression of T lymphocyte cytokines in peripheral blood of renal transplant recipients].

OBJECTIVE: To evaluate the clinical values of T-lymphocyte cytokines in renal transplant acute rejection. METHODS: A total of 31 recipients with renal transplantation and 15 healthy volunteers from January 2010 to January 2012 were enrolled and divided into acute rejection group (n = 11) and stable renal allograft function group (n = 20) according to the inclusion criteria. Peripheral blood was collected from the patients before transplantation, 1, 7, 14, 28 day after transplantation and acute rejection onset. Cytometric bead array (CBA) was used to monitor the levels of interleukin-17 (IL-17), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin(IL)-10, IL-6, IL-4 and IL-2. The associations of the changes and levels of cytokines in 3 groups were examined with Pearson correlation analysis. RESULTS: The levels of IL-17A, TNF, IL-10 and IL-2 in recipients before transplantation were (3.40 ± 1.29) , (1.79 ± 0.53) , (2.73 ± 0.65) and (1.79 ± 1.02) ng/L respectively and decreased significantly compared to healthy volunteers ((4.52 ± 2.01), (3.36 ± 1.09) , (3.91 ± 0.42) , (3.12 ± 1.07) ng/L respectively, all P < 0.05). However the levels of IFN-γ, IL-6 and IL-4 showed no significant changes between two groups (all P > 0.05). In acute rejection group after transplantation, the levels of IL-17A, IFN-γ, IL-10 and IL-6 were (9.47 ± 4.75) , (5.01 ± 2.23) , (12.20 ± 5.79) , (6.55 ± 3.45) ng/L respectively and increased significantly compared to the renal allograft function group ((4.39 ± 1.26), (2.90 ± 0.87),(5.68 ± 2.25) and (2.10 ± 0.70) ng/L respectively, all P < 0.05); the level of IL-17A was correlated with those of IFN-γ and IL-4 (Pearson r = 0.519, 0.395, both P < 0.01), the level of IFN-γ was correlated with those of IL-4 and IL-2 (r = 0.276, 0.335, all P < 0.05) , the level of TNF was correlated with that of IL-4 (r = 0.423, P < 0.05) and the level of IL-10 was correlated with that of IL-6 (r = 0.361, P < 0.05). CONCLUSIONS: Cytokines play an important role in acute rejection of renal transplant. And further understanding of its mechanism may provide experimental and preventive rationales.

[The modified extracorporeal photochemotherapy promotes apoptosis of spleen lymphocytes].

OBJECTIVE: To explore the efficacy of the modified extracorporeal photochemotherapy (ECP) in improving the apoptotic rate of lymphocytes in vitro. METHODS: The spleens which were obtained from liver transplantation donor under aseptic condition were used as experimental materials. Splenic lymphocytes (SPs) suspensions were prepared by modified and traditional ECP method, respectively. And then the isolated SPs were treated by the irradiation of 8-methoxypsoralen (8-MOP) combined with ultraviolet A (UVA) named PUVA, 8-MOP and UVA, and compared with a blank group meanwhile. The treated SPs were cultured overnight in an incubator at 37 Degrees Celsius, in a humidified atmosphere of 50 mL/L CO2 for 6-8 hours. The morphological changes of cells were observed using an inverted microscope, the apoptotic rates of SPs were detected by flow cytometry, and the difference between groups was analyzed finally. RESULTS: The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the modified ECP method were respectively (95.33±3.03)% and (97.10±2.12)% after treated by PUVA, (23.39±4.55)% and (36.32±6.63)% after treated by 8-MOP, and (66.98±3.60)% and (68.65±4.35)% by UVA. Compared with control group (12.82±1.86% and 13.4±2.65%), there were statistically significant differences (P<0.01). The apoptotic rate at early stage and the total apoptotic rate of SPs prepared by the traditional ECP method were respectively (79.73±4.21)% and (82.70±4.13)%, (61.42±2.28)% and (68.91±2.18)%, (19.30±1.78)% and (28.06±1.88)%, (10.84±0.98)% and (12.77±1.22)%, and the statistical comparisons between groups also had significant difference (P<0.01). In addition, there was a significant difference in the early and total apoptosis between the modified and traditional ECP (P<0.01), but no obvious variation in the end-stage apoptosis in the two groups (P>0.05). CONCLUSION: The modified ECP method can promote apoptosis of SPs in vitro conveniently, safely and efficiently, especially in the early stage. This can lay a foundation for the further study on dendritic cell immunomodulation induced by ECP method.

HLA-G expression in the peripheral blood of live kidney transplant recipients.

BACKGROUND: The human leukocyte antigen-G (HLA-G) has been considered to be an important tolerogeneic molecule playing an essential role in maternal-fetal tolerance, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. The aim of this study was to investigate the clinical relevance of the expression of membrane HLA-G (mHLA-G), intracellular HLA-G (iHLA-G), and soluble HLA-G (sHLA-G) in the peripheral blood of live kidney transplant recipients. METHODS: We compared the expression of the three HLA-G isoforms in three groups, healthy donors (n=20), recipients with acute rejection (n=19), and functioning transplants (n=30). Flow cytometry was used to detect the expression of mHLA-G and iHLA-G in the T lymphocytes of peripheral blood from subjects in the three groups. Enzyme-linked immunosorbent assays were used to detect sHLA-G in the plasma from the three groups. RESULTS: There were no significant differences in mHLA-G and intracellular HLA-G among the three groups, but the sHLA-G plasma level was higher in the functioning group than in the acute rejection or healthy group. We found a subset of CD4(+)HLA-G(+) and CD8(+)HLA-G(+) T lymphocytes with low rates of mHLA-G expression in the peripheral blood of kidney transplantation recipients. Intracellular expression of HLA-G was detected in T lymphocytes. However, there was no correlation between acute rejection and the mHLA-G or intracellular HLA-G expression. CONCLUSION: sHLA-G was the major isoform in the peripheral blood of live kidney transplant recipients and high sHLA-G levels were associated with allograft acceptance.

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4+CD25+Foxp3+ regulatory T cells and down-regulates cardiac allograft rejection.

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-gamma by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4(+)CD25(high)Foxp3(+) regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

Comparison of modes of prothrombin time reporting in patients with advanced liver disease associated with viral hepatitis.

BACKGROUND: Prothrombin time is widely utilized for evaluation of liver disease severity. However, its standardization of modes of reporting is not established for universal purpose. Variability in thromboplastin reagents leads to large intralaboratory and interlaboratory differences in PT results. OBJECTIVE: The aim of this study was to establish standardization of modes of PT reporting by the interchangeability analysis of prothrombin time in patients with advanced liver disease associated with viral hepatitis measured with different thromboplastin reagents by the use of various methods of expression, i.e. prothrombin time (PTs), prothrombin activity percentage (PTp), prothrombin time ratio (PTr) and International Normalized Ratio (INR). METHODS: we prospectively collected blood samples from 61 patients with advanced liver disease associated with viral hepatitis, control patients were on warfarin (n = 20). PT was measured on a STA-R with six thromboplastin reagents. PT was expressed in PTs, PTr, PTp, and INR. Neoplastin was selected as reference reagent for comparison of methods of reporting. RESULTS: The study revealed the closest agreement of the results in study population between Neoplastin and the other five reagents, and the regression lines of these reagents were close to each other, when the results were expressed in PTp while INR, PTs and PTr is not valid for comparison of patients with liver disease. In patients on oral anticoagulant therapy, only INR standardize PT results. CONCLUSION: we conclude that, in patients with liver disease, only activity percentage expression may provide a common international scale of PT reporting.

Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide.

RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system.

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.

[A discussion of standardization for prothrombin time in patients with advanced liver diseases].

OBJECTIVE: To determine which expression mode of prothrombin time (PT) might achieve PT standardization in patients with advanced liver diseases. METHODS: PT was measured with six thromboplastins with different ISI values in 16 severe chronic hepatitis patients, 50 decompensated liver cirrhosis patients and 30 patients on oral anticoagulation therapy. The results were expressed in PT (second), PTA (%), PTR and INR. RESULTS: In chronic hepatitis patients, the means of the six group's PTAs ranged from 24% to 34%, while their upper limits ranged from 47% to 61%. The means of the INRs ranged from 2.55 to 5.13, while their upper limits ranged from 4.65 to 12.77. Through one-way ANOVA of repeated measures, PPTA (0.489) was > PINR (0.120). In patients with liver cirrhosis, the means of the PTA in six groups ranged from 50% to 59%, while their upper limits ranged from 82% to 90%. The means of the INR ranged from 1.40 to 1.80, while their upper limits ranged from 1.97 to 3.69. Through one-way ANOVA of repeated measures, PPTA (0.102) was > PINR (0.01). In patients on oral coagulation therapy, the means of PTA ranged from 26% to 37%, while their upper limits ranged from 39% to 49%. The means of INR ranged from 2.35 to 2.66, while their upper limits ranged from 3.16 to 4.26. Through one-way ANOVA of repeated measures, PPTA (0.01) was less than PINR (0.102). The correlation between the results detected by Neoplastine and by other reagents were analyzed. They correlated well with each other when PTA was used as the expression mode of PT in patients with advanced liver disease. But in patients on oral anticoagulation therapy, when only the INR was used as the expression mode of PT, the correlation was well with each other. CONCLUSION: The use of INR provides inadequate standardization. Only when the PT is expressed in PTA, then it may provide a standardization mode in patients with advanced liver diseases.

[The relationship among the counts of platelet, thrombopoietin and spleen index in patients with liver cirrhosis].

OBJECTIVE: To determine the reason of thrombocytopenia in patients with liver cirrhosis, we studied the relationship among platelet counts, serum thrombopoietin (TPO) level and spleen index. METHODS: Serum TPO, platelet counts and spleen index were measured in 71 cirrhotic patients. TPO was measured with ELISA method, spleen index were measured on ultrasonography by the same doctor. RESULTS: Platelet counts in patients with cirrhosis were lower than that of healthy group [(109.20+/-53.39) vs (169.63+/-26.60) x 10(12)/L, P<0.05]. Serum thrombopoietin level in patients with cirrhosis was similar to that of healthy group [(436.42+/-258.97) vs (412.63+/-132.80) pg/ml, P>0.05]. However, serum thrombopoietin level decreased as liver disease aggravated, [(526.13+/-317.44) pg/ml in Child-Pugh grade A, (445.22+/-214.90) pg/ml in grade B and (311.45+/-182.66) pg/ml in grade C, grade A vs. Grade C, P<0.05]. However, decline in platelet counts was accompanied with incline in spleen index coordinately. 35 of 71 cirrhotic patients had normal platelet counts whereas 36 of them had thrombocytopenia. Thrombopoietin levels were higher in non-thrombocytopenia group than in thrombocytopenia group [(529.43+/-282.64) vs. (351.27+/-228.25)pg/ml, P<0.01]; but spleen index of two groups showed no difference [(29.65+/-12.00) vs. (36.35+/-12.68) cm2, P>0.05]. Correlation was found between thrombopoietin level and platelet counts (r=0.252, P=0.025); no correlation was found between spleen index and platelet counts (r=-0.238, P=0.062). CONCLUSION: The decline serum TPO levels might play an important role for thrombocytopenia in patients with liver cirrhosis.

[The contrast study of Pre-S1 protein, HBV-DNA and HBeAg in diagnosing viral replication in patients with chronic hepatitis B].

OBJECTIVE: To determine the role of Pre-S1 protein in diagnosing viral replication in patients with chronic hepatitis B. METHODS: 104 consecutive patients with chronic hepatitis B were included in the study, liver biopsy were performed in all patients. Serial serum samples were studied with the quantitative determination of HBV-DNA by a quantitative PCR assay, determination of Pre-S1 protein by ELISA. RESULTS: The positive rates of HBV-DNA and Pre-S1 protein in patients with HBsAg HBeAg anti-HBc (+) both were 96.5%. The positive rates of HBV-DNA and Pre-S1 protein in patients with HBsAg anti-HBe anti-HBc (+) were 81.5%, 72.3%, respectively. The positive rates of HBV-DNA and Pre-S1 protein in patients with HBsAg anti-HBc (+) were 87.5%, 75.0%, respectively. It represented some patients with HBeAg (-) anti-HBe (+/-) still had viral replication. HBV-DNA>10(3) copy/ml as positive criteria for diagnosing viral replication, the positive rate of HBeAg, Pre-S1 were 31.5% (28/89), 80.9% (72/89) in patients with HBV-DNA>10(3) copy/ml, respectively. The concordance rates of HBeAg, Pre-S1 with HBV-DNA were 40.0% (42/104), 82.0% (85/104), respectively. CONCLUSION: It showed that Pre-S1 was more sensitive than HBeAg in diagnosing viral replication in patients with chronic hepatitis B.