Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV.

OBJECTIVE: HBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which may have oncogenic consequences. However, the dlDNA proportion relative to total HBV DNA and its clinical significance in patients remain to be investigated. DESIGN: Based on the structural difference between rcDNA and dlDNA, we developed a peptide nucleic acid (PNA)-mediated quantitative real-time PCR (qPCR) clamping assay to measure the proportions of dlDNA in total HBV DNA in sera obtained from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) or LC-developed hepatocellular carcinoma (HCC). The factors that influence the proportion of dlDNA were also investigated. RESULTS: The average dlDNA proportion was approximately 7% in the sera of chronic HBV-infected patients and was elevated in CHB patients with abnormal levels of alanine aminotransferase. The sera dlDNA proportions increased to approximately 14% and 20% in the patients with LC and HCC, respectively. Interferon-α treatment slightly increased the dlDNA proportion in the responders; and nucleotide analogue therapy spuriously elevated the proportion. Moreover, treatment of human hepatoma cells supporting HBV replication with inflammatory cytokines significantly altered the dlDNA proportion in vitro. CONCLUSIONS: Using a novel PNA-mediated qPCR clamping assay, we first showed that serum dlDNA proportions progressively increased during the development of HBV-related liver diseases. The dlDNA proportion can be regulated by inflammatory cytokines, suggesting an association among inflammation, increased production of HBV dlDNA and development of HCC.

CD4+CXCR5+ T cells activate CD27+IgG+ B cells via IL-21 in patients with hepatitis C virus infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG+ B cells in patients with chronic hepatitis C (CHC) infection have not been thoroughly elucidated. CD4+CXCR5+ follicular helper T (Tfh) cells, via interleukin (IL)-21 secretion, activate B cells. However, the role of CD4+CXCR5+ T cells in the activation of IgG+ B cells in CHC patients is not clear. METHODS: The frequency of IgG+ B cells, including CD27-IgG+ B and CD27+IgG+ B cells, the expression of the activation markers (CD86 and CD95) in IgG+ B cells, and the percentage of circulating CD4+CXCR5+ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The concentrations of serum IL-21 were analyzed using ELISA. The role of CD4+CXCR5+ T cells in the activation of IgG+ B cells was investigated using a co-culture system. RESULTS: A significantly lower proportion of CD27+IgG+ B cells with increased expression of CD86 and CD95 was observed in CHC patients. The expression of CD95 was negatively correlated with the percentage of CD27+IgG+ B cells, and it contributed to CD27+IgG+ B cell apoptosis. Circulating CD4+CXCR5+ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4+CXCR5+ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27+IgG+ B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27+IgG+ B cells. CONCLUSIONS: HCV infection increased the frequency of CD4+CXCR5+ T cells and decreased the frequency of CD27+IgG+ B cells. CD4+CXCR5+ T cells activated CD27+IgG+ B cells via the secretion of IL-21.

[Effects of Combined Stress of Polyethylene and Sulfamethazine on Seed Germination, Seedling Growth, and Physiological Characteristics of Soybean].

The combined pollution of antibiotics adsorption by microplastics has become inevitable in soil ecosystems; moreover, the plant biological effects under combined stress remain unclear. This study used soybean variety Jindou 21 as the material and conducted seed germination test and soil-potted seedling experiment to study the effects of different single and combined treatments of polyethylene (PE) and sulfamethazine (SMZ) on seed germination, seedling growth, photosynthetic parameters, chlorophyll fluorescence parameters, and nitrogen metabolism. The results showed that single PE treatment at low levels promoted soybean seed germination and seedling growth physiology; however, inhibited them at a high level. A lower-level PE treatment[10 mg·L-1 (or mg·kg-1)] could promote soybean seed germination, seedling growth, photosynthesis, and nitrogen metabolism, whereas a higher level PE treatment[100 mg·L-1 and 200 mg·L-1 (or mg·kg-1)] had significant inhibition. The single SMZ treatment had different degrees of inhibition on soybean seed germination and seedling growth physiology, and the inhibition degree increased with the increase in SMZ treatment level. Under the different levels of combined treatments of PE and SMZ, adding the lower level PE treatment could alleviate the inhibition of the single SMZ treatment on soybean, with 10 mg·L-1(or mg·kg-1) PE+1 mg·L-1(or mg·kg-1) SMZ treatment having the best comprehensive mitigation effect, which could increase soybean seed germination potential, germination rate, germination index, vigor index, plant height, root length, shoot and root fresh weight, Pn, Gs, Tr, chlorophyll contents, Fv/Fm, ΦPSⅡ, ETR, qP, and key enzyme activities for nitrogen metabolism such as NR and decrease the average germination time, Ci, NPQ, and NO3--N and NH4+-N contents compared with those in the single SMZ treatment. Adding the higher level PE treatment enhanced the inhibition of SMZ on soybean, and the inhibition degree increased with the increase in SMZ treatment level, in which 200 mg·L-1(or mg·kg-1) PE+50 mg·L-1(or mg·kg-1) SMZ treatment yielded the greatest inhibition. In summary, the lower level PE treatment could alleviate the inhibition of SMZ on soybean seeds and seedlings to a certain extent; however, the higher level PE treatment could produce a synergistic effect with SMZ, thus aggravating the toxic effect of the single stress treatment.

[Preliminary study of estrogen effects on calcium free smooth muscle cells at the endometrial-myometrial interface in uteri with adenomyosis].

OBJECTIVE: To investigate the effect and mechanism of estrodial (E(2)) on intracellular free calcium in the endometrial-myometrial interface (EMI) smooth muscle cells from uteri with adenomyosis. METHODS: From March 2011 to October 2011, 16 uterus specimens were collected from patients with adenomyosis undergoing hysterectomy in Beijing Obstetrics and Gynecology Hospital, which included 9 proliferative endometrium and 7 secretory endometrium. EMI smooth muscle cells from the uterus were cultured and loaded with calcium ion (Ca(2+)) fluorescent probe fluo-4/AM. The labeled cells were stimulated with the various concentration of E(2)(1×10(2), 1×10(3), 1×10(4), 1×10(5) pmol/L, respectively), then the changes of intracellular Ca(2+) fluorescence intensity were measured by laser scanning microscopy. The most suitable concentration of E(2) was selected, and the reaction difference between the EMI smooth muscle cells of two menstrual phases were also investigated; The changes of intracellular Ca(2+) fluorescence intensity were detected proliferative and secretory smooth muscle cells in E(2) conjugated to bovine serum albumin (17ß-E(2)-BSA) group, cycloheximide (CHX) group, fulvestrant (ICI182780) group and pertussis toxin (PTX) group. RESULTS: (1) The cell viability of primary cultured EMI smooth muscle cells was well at 24 hours culture. (2) 1×10(2) - 1×10(5) pmol/L E(2) can rapidly increase the intracellular Ca(2+) fluorescence intensity within 1 min (P < 0.01);The increased amplitudes caused by 1×10(4) pmol/L and 1×10(5) pmol/L E(2) were the most significant, but there was no significant difference between them (P > 0.05). 1×10(4) pmol/L was the most suitable concentration. (3) With the 1×10(4) pmol/L E(2), the Ca(2+) fluorescence intensity changes showed no significant difference between the EMI smooth muscle cells from the proliferative phase and secretory phase uterus (P > 0.05). The Ca(2+) fluorescence intensity changes were 646 ± 32 in 17ß-E(2)-BSA group and 602 ± 31 in CHX group, when compared with 513 ± 26 and 617 ± 35 in respective control group, no significant difference was observed (P > 0.05). The increased amplitude of 188 ± 20 in the PTX group and 302 ± 11 in ICI182780 group exhibited significant difference with 632 ± 33 and 635 ± 24 in respective control group (P < 0.01). CONCLUSION: E(2) could increase the intracellular Ca(2+) of EMI through a membrane receptor dependent and nongenomic mechanism of action.

[Correlation between interleukin-28B genetic polymorphisms and primary hepatocellular carcinoma].

OBJECTIVE: To explore the correlation between single nucleotide polymorphisms (SNPs) of interleukin-28B (IL-28B) gene and the susceptibility to primary hepatocellular carcinoma (HCC). METHODS: A total of 300 histologically confirmed HCC cases (from November 2001 to April 2010) and 310 healthy controls with no history of chronic hepatitis B or hepatocellular carcinoma (2009-2010) were selected from a hospital in Guilin and a hospital in Beijing for this case-control study.139 HCC patients in the case group had complete clinical tracking data. All the subjects were Han Chinese, with no age or gender restrictions.2 ml peripheral blood samples were drawn from each subject with informed consent. SNP of rs12972991, rs4803223, rs8099917 and rs12979860 four loci in IL-28B gene were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). RESULTS: The frequencies of C allele at rs12972991, G allele at rs8099917 and G allele at rs4803223 were 6.7% (40/598), 7.9% (47/598) and 10.0% (59/588) respectively in case group; all higher than the corresponding frequencies in control group, separately 2.9% (18/618), 4.1% (25/616) and 3.6% (21/608). The differences were statistically significant (χ2=9.542, 7.858, 20.736, P values all<0.05). The above alleles could increase the risk of HCC, and the OR (95%CI) values were separately 1.67 (1.13-2.46), 1.49 (1.08-2.06) and 2.91 (1.79-4.72). The genotype frequencies of AC+CC at rs12972991, GT+GG at rs8099917, GA+GG at rs4803223 were 13.0% (39/299), 14.7% (44/299) and 19.0% (56/296) respectively in case group; while the frequencies were lower in control group, separately 5.8% (18/309), 8.1% (25/308) and 6.6% (20/304). The differences were statistically significant (χ2=9.319, 6.557, 20.948, P values all<0.05). These genotypes may increase the risk of HCC, and the adjusted OR (95%CI) values were 2.24 (1.31-3.83), 1.81 (1.14-2.88) and 2.90 (1.78-4.70), respectively. The stratified analysis of the clinical data indicated that the frequency of genotype GA+GG at rs4803223 was 50.0% (13/26) in patients of tumor thrombosis in portal vein (TTPV), higher than the frequency of genotype AA (21.1%, 23/109). The difference was statistically significant (χ2=8.965, P=0.003). CONCLUSION: The results suggested that IL-28B gene polymorphisms was correlated to the susceptibility to HCC in Chinese Han ethnic population. Among them, GA + GG genotype at rs4803223 could increase the risk of TTPV in HCC patients.

PD-1/PD-L1 combined with LAG3 is associated with clinical activity of immune checkpoint inhibitors in metastatic primary pulmonary lymphoepithelioma-like carcinoma.

Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is an Epstein-Barr virus (EBV)-related, rare subtype of non-small-cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICI) show durable responses in advanced NSCLC. However, their effects and predictive biomarkers in PLELC remain poorly understood. We retrospectively analyzed the data of 48 metastatic PLELC patients treated with ICI. Pretreated paraffin-embedded specimens (n = 19) were stained for PD-1, PD-L1, LAG3, TIM3, CD3, CD4, CD8, CD68, FOXP3, and cytokeratin (CK) by multiple immunohistochemistry (mIHC). Next-generation sequencing was performed for 33 PLELC samples. Among patients treated with ICI monotherapy (n = 30), the objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), and overall survival (mOS) were 13.3%, 80.0%, 7.7 months, and 24.9 months, respectively. Patients with PD-L1 ≥1% showed a longer PFS (8.4 vs. 2.1 months, p = 0.015) relative to those with PD-L1 <1%. Among patients treated with ICI combination therapy (n = 18), ORR, DCR, mPFS, and mOS were 27.8%, 100.0%, 10.1 months, and 19.7 months, respectively. Patients with PD-L1 ≥1% showed a significantly superior OS than those with PD-L1 <1% (NA versus 11.7 months, p = 0.001). Among the 19 mIHC patients, those with high PD-1/PD-L1 and LAG3 expression showed a longer PFS (19.0 vs. 3.9 months, p = 0.003). ICI also showed promising efficacy for treating metastatic PLELC. PD-L1 may be both predictive of ICI treatment efficacy and prognostic for survival in PLELC. PD-1/PD-L1 combined with LAG3 may serve as a predictor of ICI treatment effectiveness in PLELC. Larger and prospective trials are warranted to validate both ICI activity and predictive biomarkers in PLELC. This study was partly presented as a poster at the IASLC 20th World Conference on Lung Cancer 2019, 7-10 September 2019, Barcelona, Spain.

High SHP2 expression determines the efficacy of PD-1/PD-L1 inhibitors in advanced KRAS mutant non-small cell lung cancer.

BACKGROUND: Src homology region 2 domain-containing phosphatase 2 (SHP2) is a novel target for Kirsten rat sarcoma oncogene (KRAS) mutant cancer. We retrospectively studied the significance of SHP2 in KRAS mutant non-small cell lung cancer (NSCLC) treated with immunotherapy and its relationship with tumor microenvironment (TME). METHODS: Sixty-one advanced KRAS mutant NSCLC patients who underwent immunotherapy were enrolled. Next-generation sequencing (NGS) was used to profile mutation status. The expression of SHP2, phospho-SHP2 (pSHP2), and programmed death ligand 1 (PD-L1) were analyzed by immunohistochemistry (IHC). Quantitative multiplexed immunofluorescence cytochemistry (mIFC) analysis was conducted to describe the TME. RESULTS: SHP2 was heterogeneously expressed in 32 samples in both tumor cells and immune cells and highly expressed (H-score >10) in 25 (78.1%) samples. The expression levels of SHP2 and pSHP2 were positively correlated. Stromal SHP2 (s-SHP2) was higher in tumors with PD-L1 ≥50% versus PD-L1 <50% (p = 0.039). By quantitative mIFC analysis, the expression of s-SHP2 had positive correlation with CD8, CD4, CD68, and PD-L1 levels in stromal area. Patients with high SHP2 expression made up 100.0% of the partial respond (PR) and 80.0% of the stable disease (SD), whereas 50.0% of the progress disease (PD). High SHP2 expression was associated with longer progression-free survival (PFS) and overall survival (OS) (p < 0.001, p = 0.013). Patients with high expression of both SHP2 and PD-L1 had longer PFS (p < 0.001). CONCLUSION: High SHP2 expression could predict the efficacy of immunotherapy and better survival in advanced KRAS mutant NSCLC. SHP2 may function in both tumor cells and immune cells, warranting further study on the potential diverse effects of SHP2 inhibition in TME.

[Expression of ERα in endometrial-myometria interface of human adenomyosis].

OBJECTIVE: To examine and analyze the expression of estrogen receptor-α (ERα) in myometrium of endometrial-myometria interface (EMI) of adenomyosis and normal myometrium and explore the pathogenesis of adenomyosis (ADS). METHODS: The myometrium specimens were obtained from 41 cases undergoing hysterectomy, including 20 adenomyosis patients in ADS group and 21 other patients in control group. EMI was located and acquired by anatomy and immunohistochemistry. The EMI smooth muscle cells were isolated immediately post-operatively for primary culture. RT-PCR and Western blot were used to detect ERα in myometrium of EMI in two groups. RESULTS: (1) Uterine smooth muscle cells of EMI were in an excellent condition and cell viability was fair; (2) the expression of ERα in myometrium of EMI of ADS group showed no cyclic change. While in myometrium of EMI in control group, it showed obvious cyclic change. The expression level was significantly higher in proliferative stage than that in secretory stage (P < 0.05); (3) there was no significant difference in the expression of ERα between the ADS (0.18 ± 0.023) and control groups (0.19 ± 0.024) during the proliferative phase. During the secretory phase, it showed significant difference in ERα expression. And the ADS group (0.17 ± 0.032) was much higher than the control group (0.12 ± 0.015) (P > 0.05). CONCLUSION: The loss of periodic expression of ERα in myometrium of EMI of adenomyosis may be associated with an abnormal regulation of estrogen in adenomyosis.

Expression of CIITA-related MHCII molecules in tumors linked to prognosis in hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) after surgery is poor due to its high recurrence rate. In order to unfold the mechanism of different recurrent-free survival (RFS) times following resection, expression profiling of tumor tissues from 32 HCC patients with different RFS time were used to identify differential expression of individual genes and signaling pathway components correlated with RFS time. Quantitative RT-PCR, Western blotting, and immunohistochemistry were used to validate the expression of selected genes. Up-regulation of several immune related genes and pathways, especially HLA II-related antigen presenting pathways, significantly correlated with longer RFS time. The expression of MHCII molecules were found to be mainly located in either CD68+ cells or CD45+ cells, and their expression significantly correlated with the expression of CIITA (HLA II genes transactivator) in the tumor. The results suggest that the high expression level of CIITA and MHCII molecules in hepatocellular carcinoma tissue is an effective prognostic marker for longer RFS time in HCC.

Comparisons for detecting NY-ESO-1 mRNA expression levels in hepatocellular carcinoma tissues.

NY-ESO-1 is a cancer/testis (CT) antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. We compared the expression of NY-ESO-1 mRNA in hepatocellular carcinoma (HCC) patients by using various primers and DNA polymerases to optimize RT-PCR conditions and to evaluate the correlations among the expression levels of NY-ESO-1, LAGE-1 and SSX-1 and clinical parameters. We determined differences in the abilities of the various primers and DNA polymerases to amplify the NY-ESO-1 gene at different exons. Primers designated as P3 detected targeted sequences better than primers P1 and P2; AmpliTaq Gold DNA polymerase was more effective than Platinum pfx DNA polymerase and Taq DNA polymerase. NY-ESO-1, LAGE-1 and SSX-1 mRNAs were detected in 29.7, 45.3 and 37.5%, respectively, of the 64 HCC specimens. No CT antigen mRNAs were detected in the 64-paired adjacent non-cancerous tissues. The frequency for the co-expression of one, two or three antigens of NY-ESO-1, LAGE-1 and SSX-1 was 57.8, 35.9 and 18.8%, respectively. We also analyzed the relationships among the CT antigen expression levels and several clinical parameters. There were no significant differences between CT antigen expression levels and clinical parameters, except the correlations between the expression of SSX-1 and the age of the patients.

[Specific suppression in regulatory T cells by Foxp3 siRNA contributes to enhance the in vitro anti-tumor immune response in hepatocellular carcinoma patients].

OBJECTIVE: To design the suitable sequence siRNA of Foxp3 to interfere the function of regulatory T cells(Treg), and to evaluate whether the suppression of Treg could enhance the anti-tumor immune response in hepatocellular carcinoma patients or not. METHODS: Foxp3-specific siRNAs by chemical synthesis were delivered into regulatory T cells. The inhibition efficiencies of Foxp3-specific siRNAs were evaluated by real-time PCR and fluorescently stained for intracellular Foxp3 and analyzed using multiparameter FCM. The Foxp3(+) Treg subpopulation was selectively analyzed for surface expression levels of CD127, CTLA-4 and GITR. The suppression of Treg to CD4(+)CD25(-) T cells or anti-tumor specific CD8(+) T-cell responses induced by tumor specific antigen (NY-ESO-1b) was evaluated by CFSE, Elispot and Pentamer analysis. RESULTS: A subpopulation of Tregs with reduced levels of Foxp3 mRNA and protein mediated by siRNA was CD127 up-regulation and CTLA-4 or GITR down-regulation compared with those in Foxp3(high) Tregs. Knockdown of intracellular Foxp3 in Treg reduced suppression of Foxp3+ Treg to proliferative capacity of CD4(+)CD25(-) T cells. IFN-gamma released of NY-ESO-1-specific CD8(+) T cells from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected(132+/-55 vs 27+/-11, P<0.05). Frequency of NY-ESO-1b-specific CD8(+) T cells by Pentamer analysis from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected (0.21%+/-0.17% vs 0.57%+/-0.39%, P<0.05). CONCLUSION: Knockdown of intracellular Foxp3 in Treg mediated by siRNAs can inhibit the suppression of Foxp3+ Treg and enhance the anti-tumor immune response in hepatocellular carcinoma patients in vitro.

Pelvic incidence measurement using a computed tomography data-based three-dimensional pelvic model.

OBJECTIVES: To introduce a new method of pelvic incidence (PI) measurement based on three-dimensional (3D) pelvic models reconstructed from CT images and to report the normal distribution of PI in normal pelvic anatomy. METHODS: CT images of 320 subjects with normal pelvic anatomy who visited the Radiology Department between 2006 and 2017 were retrospectively selected and saved in Digital Imaging and Communications in Medicine (DICOM) format. A computerized method was employed to determine the bony landmarks required for the measurement of PI. To quantify the method's accuracy and reliability, the intraclass correlation coefficient (ICC) was calculated. A subgroup of 30 DICOM files was randomly selected to perform a validation study. Three independent testers performed all procedures. All measurements were performed twice independently by the three testers on all 10 subjects with an interval of 2 weeks. Independent samples t tests were used to identify statistically significant differences in the PI value between sexes. Pearson correlation coefficient was employed to determine the relationship between PI and age. RESULTS: PI measurement using the new method resulted in an excellent intraobserver reliability (0.9612, range 0.8917-0.9893; p < 0.001) and interobserver reliability (0.9867, range 0.9611-0.9964; p < 0.001). PI was significantly different between sexes, with larger PI in women (p = 0.019). PI was significantly larger in the 40-80-year age group (45.94 ± 9.08°) than the < 40-year age group (43.50 ± 7.39°). We did not find any linear correlation between PI and age in the male (r = 0.140, p = 0.105) or female subgroup (r = 0.119, p = 0.107). A weak correlation between PI and age overall was observed (r = 0.142, p = 0.011). CONCLUSION: Accurate PI measurement could be achieved by a CT data-based 3D pelvic model.

[Inhibition of CD4+ CD25+ regulatory T cells in chronic hepatitis B patients].

OBJECTIVE: To evaluate the inhibition of CD4+ CD25+ regulatory T cells (Treg) in the chronic hepatitis B patients. METHODS: Peripheral blood samples were collected from 22 patients with chronic hepatitis B (CHB) and 18 healthy blood donors to isolate the peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze the proportion of CD4+ CD127(lo)CD25(hi-int) Tregs in the CD4+ T cells so as to calculate the proportion of CD4+ CD25+ Tregs in the CD4+ T cells. BrdU incorporation method was used to evaluate the immune inhibition of the CD4+ CD25+ Tregs. CD4+ CD25- cells were isolated by magnetic bead sorting technique. The CD4- T cells and CD4+ CD25- T cells ere mixed and stimulated by HBVcore 18-27 peptide. The PBMCs of the CHB patients with the Treg depleted and Treg not depleted underwent detection of HBVcore18-27 specific cytotoxic T lymphocytes (CTLs). The IFN-gamma secretion of the CTLs in the PBMCs of CHB patients with Treg depleted and Treg not depleted was detected by HLA-pentamer and enzyme-linked immunospot assay (Elispot). RESULTS: The proportion of CD4+ CD127(lo)CD25(hi-int) Treg in the CD4+ T cells used to reflect the percentage of CD4+CD25+ Tregs in the CD4+ T cells of the CHB patients was 4.3% +/- 2.4%, significantly higher than that of the healthy controls (2.1% +/- 1.3%, t = 3.74, P <0.01). There was no significant difference in the inhibition of CD4+ CD25- T cells by autogenous CD4+ CD25+ T cells between the CHB patients and healthy controls. The frequency of CTLs induced by HBV core 18-27 of the CHB patients with their CD4+ CD25+ cells in circulation depleted was 0.74% +/- 0.31%, significantly higher than that of the patients whose CD4+ CD25+ cells in circulation were not depleted (0.17% +/- 0.08%, t = 4.75, P <0.01). The frequency of IFN-gamma secreting spots of HBVcore18-27-specific CD8+ T cells of the CHB patients with their CD4+ CD25+ cells depleted was (112 +/- 33), significantly higher than that of the CHB patients whose CD4+ CD25+ cells in circulation were not depleted [(23 +/- 14), t =7.828, P<0.01)]. CONCLUSION: The proportion of CD4+ CD25+ Treg in CHB patients is increased compared to the healthy blood donor. The proliferative capacity of CD4+ CD25- T cells is inhibited by the presence of CD4+ CD25+ Treg dose-dependently, and the inhibition of CD4+ CD25+ Tregs in the CHB patients is similar to the inhibition of CD4+ CD25+ Tregs in healthy donors. The elimination of Treg cells followed by stimulation with HBVcore18-27 peptide significantly improves the antivirus CTL responses in CHB patients.

The functional evaluation of dendritic cell vaccines based on different hepatitis C virus nonstructural genes.

Hepatitis C virus (HCV) nonstructural (NS) genes are relatively conserved and play critical roles in cellular immune responses against HCV. The aim of the study was to evaluate the immunogenicity of the different HCV NS genes through transduction of DCs and presentation to T cells. Monocyte-derived DCs from healthy donors were infected with the recombinant adenovirus (Ad) harboring HCV NS3 (AdNS3), NS4 (NS4A and NS4B; AdNS4), NS5 (NS5A and NS5B; AdNS5), NS3/NS4 (AdNS3/NS4), and NS4/NS5 (AdNS4/NS5) genes, and then used to stimulate autologous lymphocytes in vitro. Antigen-specific cellular immune responses were detected by interferon-gamma (IFN-gamma), interleukin 4 (IL-4), and Granzyme B (GrB) enzyme-linked immunospot assays (ELISPOT). DCs expressing different HCV NS genes all induced positive immune responses. Furthermore, DCs transfected with AdNS3/NS4 were superior to DCs infected with AdNS3 or AdNS4 in inducing HCV-specific immunity. The same results were obtained when we compared DCs infected with AdNS4/NS5 to AdNS4 or AdNS5. DCs transduced with NS3/NS4 or NS4/NS5 had similar ability to elicit specific immune responses to HCV.

[The frequency, phenotypes and functions of CD4+ CD25+ regulatory T cells in hepatocellular carcinoma patients].

OBJECTIVES: (1) To evaluate the prevalence, phenotypes and suppressive function of CD4+CD25+ regulatory T cells (Tregs) among the in peripheral blood mononuclear cells (PBMCs) and tumor-infiltration lymphocytes (TILs) from hepatocellular carcinoma (HCC) patients and patients with chronic hepatitis B. (2) To investigate the correlation between the frequency of CD4+CD25+ Tregs and clinical characteristics of HCC patients. METHODS: PBMCs and TILs in 18 HCC patients, 10 chronic hepatitis B (CHB) patients and 15 healthy donors were evaluated for the phenotypes of CD4+CD25+ Tregs and the proportion of CD4+CD25+ Tregs as a percentage of the total CD4+ cells, by flow cytometric analysis with three or four color staining. The relationship between the frequency of CD4+CD25+ Tregs and tumor TNM stages was analyzed. The CD4+CD25+ Tregs and CD4+CD25- T cells were isolated from PBMC of HCC patients and donors. The suppressive function of CD4+CD25+ Tregs was analyzed. RESULTS: The percentages of CD4+CD25+ Tregs of the HCC patients (6.38% +/- 6.30%) and CHB patients (4.29% +/- 1.82%) were significantly higher than those of the healthy donors (1.58% +/- 0.55%, P less than 0.01). Among the TILs, the percentage of CD4+CD25+ Tregs was higher (t = 4.39, P < 0.01). There were significant differences in the prevalence of CD4+CD25+ Tregs in early and advanced stage HCCs (stage II vs. III, P less than 0.05; stage II vs. IV P < 0.01). The proliferative capacity of CD4+CD25- T cells was inhibited by the presence of CD4+CD25+ T cells in a dose-dependent manner where the level of suppression was correlated to the ratio of the two-cell populations. CONCLUSION: These results suggest that the increase in frequency of CD4+CD25+ Tregs might play a role in the suppression of the immune response against HCC, which may contribute to the HCC cells that escaped from immunological surveillance.

Integrated Data Set of microRNAs and mRNAs Involved in Severe Intrauterine Adhesion.

BACKGROUND: Adhesion tissue is formed following injury to the uterine basal layer. Currently, there is no effective treatment for severe intrauterine adhesion (IUA), which causes loss of reproductive function. Enhanced understanding of the molecular mechanisms driving severe IUA would be beneficial for the treatment. METHODS: Differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in severe IUA (n = 3) and normal (n = 3) endometrium were analyzed by high-throughput microarray analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and found to overlap with the differentially expressed mRNAs. Gene Ontology and pathway analyses were performed for the intersecting genes. Three of the significantly dysregulated miRNAs and 4 of their target mRNAs were further assessed using quantitative real-time polymerase chain reaction (PCR) in 10 severe IUA and 10 normal endometrium samples. RESULTS: Microarray analysis indicated that 26 miRNAs and 1180 mRNAs were significantly different between the 2 groups. Of these, 16 miRNAs and 54 mRNAs overlapped with putative miRNA target genes and prediction of target gene. Real-time PCR revealed upregulation of hsa-miR-513a-5p and has-miR-135a-3p and downregulation of hsa-miR-543 and their corresponding target genes, plus downregulation of ADAM9 (a disintegrin-containing and metalloproteinases) and lysyl oxidase and upregulation of CDH2 (N-cadherin) and COL16A1 (collagen 16A1). Both CDH2 and COL16A1 were bioinformatically predicted and confirmed in vitro as target genes of miR-543. CONCLUSION: This study provides an integrated data set of the miRNA and mRNA profiles in severe IUA, showing involvement of many miRNAs and their target genes. Further analysis of these genes will help in understanding of the molecular mechanism of IUA formation.

[The effect of stimulation of dendritic cell on HBV-epitopic cytotoxic T-lymphocyte].

OBJECTIVE: To observe the enhancement of cytotoxic T lymphocyte responses in patients with chronic hepatitis B following vaccination with dendritic cell stimulated by Poly (I:C) in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation on Ficoll-Hypaque, the adherent cells were cultured in the medium AIM-V contained recombinant human IL-4 and recombinant human GM-CSF. On day 7, one part of wells were added with Poly (I:C). On day 9, mature DCs (mDCs) were harvested and used to phenotype analysis. Both of the immature DCs and mature DCs were concultured with the autologous T cells for another two-three days. According to the source of dendritic cells concultured with the T cells, the subjects were divided into three groups: the T cells isolated from CHB group; the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group; the T cells stimulated by dendritic cells concultured with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide group. The function and frequency of HBV specific CTL were detected by Elispot (Enzyme-linked immunospot assay, Elispot) and tetramer staining. RESULTS: The average of percentage of HBV specific CD8(+) cells of total CD8(+) cells was 0.77% (0.45% approximately 1.74%) in the T cells isolated from 22 chronic hepatitis B patients and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 16 (9 approximately 28); The percentage of HBV specific CD8(+) cells of the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group rose to 1.92% (1.36% approximately 2.65%) and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 46 (30 approximately 67); while stimulated by dendritic cells added with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide, the percentage of HBV specific CD8(+) cells of total CD8(+) cells rose to 3.49% (2.02% approximately 4.60%) and the average of the spots was 98 (59 approximately 130). There was statistical difference between the results of Elispot and tetramer of the three groups (P < 0.01). CONCLUSION: The T lymphocyte of patients with chronic hepatitis B stimulated by the autologous dendritic cells may result in obtaining high percentage and functional antigen-specific T lymphocyte. The addition of Poly (I:C) which was used as maturation promoting factor in DC culture can enhance the function of DC significantly and can get even higher percentage antigen-specific T lymphocyte with enhanced function.

[Study of phenotypes and functions of cord blood dendritic cells from fetuses whose mothers had chronic hepatitis B].

OBJECTIVE: To investigate the phenotypes and functions of cord blood dendritic cells of fetuses whose mothers are patients with chronic hepatitis B. METHODS: Peripheral blood and cord blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation with Ficoll-Hypaque. The adherent cells were cultured in AIM-V medium containing recombinant human IL-4, TNF-alpha and GM-CSF. On day 9, mature DCs (mDC) were harvested and used for phenotype analysis. The amounts of IL-12 which dendritic cells produced were measured. The dendritic cells that were studied and compared were from cord blood of fetuses of both CHB positive and negative mothers and from CHC adult peripheral blood. RESULTS: The expression rate of CD80 and CD83 of chronic hepatitis B mother cord blood dendritic cells was low compared with that of the healthy cord blood, healthy adult peripheral blood, and chronic hepatitis B adult peripheral blood, P < 0.05. The amount of IL-12 produced by chronic hepatitis B mother cord blood dendritic cells was lower than that of healthy cord blood, healthy adult peripheral blood, chronic hepatitis B adult peripheral blood (P < 0.05). The T lymphocyte proliferation inducing ability of dendritic cells of healthy adult peripheral blood was higher in inducing cord blood T lymphocytes proliferation, which was greater than that of the healthy adult peripheral blood in inducing adult T lymphocytes and was greater than that of the healthy cord blood dendritic cells in inducing cord blood T lymphocytes, which was greater than that of the healthy cord blood in inducing adult T lymphocytes, which was greater than that of chronic hepatitis B mothers in inducing cord blood T lymphocytes, which was greater than that of chronic hepatitis B mother cord blood in inducing adult T lymphocytes. CONCLUSION: The maturation and functioning of CHB mother cord blood dendritic cells were lower than those of healthy cord blood, healthy adult peripheral blood and CHB adult peripheral blood.

[Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection].

OBJECTIVE: To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy. METHODS: Recombinant class I HLA-A2 heavy chains and beta-2 M were produced in Escherichia coli cells transformed with pBV220 vectors. Only the extracellular domain of class I heavy chain was expressed, following modification by replacement of the C-terminal domain with a substrate sequence for BirA biotinylation. HLA-A2-BSP was folded in the presence of beta-2 microglobulin and a specific peptide to form a peptide-MHC complex. The MHC complexes were biotinylated using purified BirA enzyme. Biotinylated MHC-peptide complexes were purified. Tetramers were generated by mixing biotinylated protein complex with streptavidin-PE at a molar ratio of 4:1. Then analysis of stained PBMCs was performed using FACScan and CellQuest software. RESULTS: The expression levels of pBV220-HLA-A2-BSP and beta-2M were 46% and 48% of total bacterial proteins estimated from SDS - PAGE, respectively. And they were mainly located in the insoluble fraction of the cell as inclusion bodies and the proportion were about 85% and 90%, respectively. The purity of pBV220-HLA-A2-BSP and beta-2M was above 95% analyzed by SDS-PAGE, and the concentration of pBV220-HLA-A2-BSP and beta-2M was about 1.5 g/L and 1.2 g/L, respectively. Using the constructed HLA-A2-peptide tetramer to detect the HBV/HCV-specific CTL, the HBV-specific CD8(+) frequencies were 1.84% and 0.02% - 0.68% of the total CD8(+) T cells in acute and chronic HBV hepatitis, respectively. As an additional control, an HLA-A2/HCV tetramer was tested in the acute and chronic HBV hepatitis. The frequencies never exceeded 0.02% of the total CD8(+) T cell number. Similar low levels of background staining were also detected in the HLA-A2(+) or A2(-) healthy control. The HCV-specific CD8(+) frequency was 0.02 - 0.72% of the total CD8(+) T cells in chronic HCV hepatitis. The same frequencies of control were detected. CONCLUSION: High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs. Especially, using these two HBV and HCV-specific tetramer can detect the frequencies of the HBV/HCV-specific CD8(+) T cells directly in vitro.

Protein-losing enteropathy in systemic lupus erythematosus: 12 years experience from a Chinese academic center.

OBJECTIVE: Protein-losing enteropathy (PLE) is a complication in some systemic lupus erythematosus (SLE) patients that is often misdiagnosed. With this study, we provide insight into clinical characteristics, laboratory characteristics, diagnostic tests, risk factors, treatment, and prognosis of the disease. METHODS: A retrospective, case-control study was performed in 44 patients with SLE-related PLE (PLE group) and 88 patients with active SLE (control group) admitted to our care from January 2000-January 2012. Risk factors for SLE-related PLE were examined, and we analyzed the accuracy of single and combined laboratory characteristics in discriminating SLE-related PLE from active SLE. Serum albumin and C3 levels were measured as outcome during and after treatment with corticosteroids and immunosuppressive agents. RESULTS: The PLE group had lower mean serum albumin and 24-hour urine protein levels, higher mean total plasma cholesterol levels, and greater frequencies of anti-SSA and SSB seropositivity compared with the control group. Anti-SSA seropositivity, hypoalbuminemia, and hypercholesterolemia were independent risk factors for SLE-related PLE. The simultaneous presence of serum albumin (<22 g/l) and 24-hour urine protein (<0.8 g/24 h) had high specificity, positive predictive value, negative predictive value, and positive likelihood ratio, a low negative likelihood ratio and no significant reduction in sensitivity. High dosage of glucocorticosteroid combined with cyclophosphomide were mostly prescribed for SLE-related PLE. CONCLUSION: SLE-related PLE should be considered when an SLE patient presents with generalized edema, anti-SSA antibody seropositivity, hypercholesterolemia, severe hypoalbuminemia, and low 24-hour urine protein levels. Aggressive treatment for lupus might improve prognosis.