[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.

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.

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.

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.

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.

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.

In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells: interaction between EPCs and HSCs.

Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability.

[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.

[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.

In vitro inhibition of HBV replication by a novel compound, GLS4, and its efficacy against adefovir-dipivoxil-resistant HBV mutations.

BACKGROUND: HBV infection continues to be an important worldwide cause of morbidity and mortality. Patients with chronic hepatitis B can be successfully treated using nucleoside/nucleotide analogues. However, drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease. Here, we report the effects of GLS4, a non-nucleosidic inhibitor that exhibits a novel and highly specific anti-HBV activity. METHODS: The median inhibitory concentrations (IC(50)s) of GLS4 on HBV were measured by Southern blotting. HBV capsid and core protein levels were detected by immunoblotting. To determine the antiviral activity of GLS4 against adefovir dipivoxil (ADV)-resistant HBV mutants, HepG2 cells transiently transfected with PUC-HBV1.2 plasmids that contained one of three major ADV-resistant mutations (rtA181T, rtA181V and rtN236T) were treated with GLS4. Intracellular HBV replicative intermediates were detected by Southern blotting. The effect on the in vitro assembly of HBV capsid protein was examined using dynamic light scattering and electron microscopy. RESULTS: The IC(50) of GLS4 was 0.012 µM, which is significantly lower than that of lamivudine (0.325 µM). Immunoblot analysis of HepG2.2.15 cells and transiently transfected HepG2 cells indicated that GLS4 treatment interfered with the formation of core particles (assembly). The ADV-resistant HBV mutant strains were also sensitive to GLS4. Upon examining the in vitro assembly of HBV core protein 149 by electron microscopy, increased aberrant particles were observed after GLS4 treatment. CONCLUSIONS: GLS4 is a new and unique potential anti-HBV agent that possesses a different mechanism of action than existing therapeutic drugs.

Differences in gene expression profiles and carcinogenesis pathways between colon and rectal cancer.

OBJECTIVE: Colon cancer is more common in the USA and Europe than that in China, for reasons that are unclear. The aim of this study was to investigate the differences in gene expression profiles and carcinogenesis pathways between colon and rectal cancer. METHODS: Expression profiling of primary tumor tissues from 12 colon and 12 rectal cancers was performed using oligonucleotide microarray analysis. All samples were strictly matched by clinical features. Bioinformatic analyses such as the Kyoto Encyclopedia of Genes and Genomes were used to identify genes and pathways specifically associated with colon or rectal cancers. RESULTS: A total of 824 genes were differentially expressed in colon and rectal cancers. All differential gene interactions in the Signal-Net were analyzed. More genes were differentially expressed and included in the Signal-Net for rectal than colon cancer. Of the genes differentially expressed between colon and rectal cancer, S100P, the Reg family, ACTN1, CAMK2G and ACAT1 were the most significantly altered. Genes involved in the cell cycle pathway were present in rectal and colon cancers, but were more important in rectal cancer. The p53 and metabolic signaling pathways were significantly different in colon and rectal cancers. Gene expression profiles differed between colon and rectal cancer, with metabolic pathways being more important in rectal cancer. CONCLUSION: The oncogenesis of rectal cancer may be more complex than that of colon cancer. Some genes could be new biomarkers for distinguishing between these two cancers.

[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.

Regulatory T cell depletion enhances tumor specific CD8 T-cell responses, elicited by tumor antigen NY-ESO-1b in hepatocellular carcinoma patients, in vitro.

Immunotherapy in hepatocellular carcinoma based on one or a few tumor specific antigens have shown limited antitumor efficacy. As a major suppressive factor in tumor immune response, better understanding of the role of regulatory T cells (Tregs) in hepatocellular carcinoma might be important for design of future immunotherapy-based clinical protocols. Tregs from 49 HCC patients and 40 controls were identified by flow cytometric analysis for the phenotype. Functional studies were performed by analyzing their inhibition to immune responses. Finally investigating whether elimination of Tregs was capable of enhancing the immunostimulatory efficacy of NY-ESO-1b peptides. In HCC peripheral blood and tumor-infiltrating lymphocytes, we found increased numbers of Tregs, which expressed high levels of HLA-DR, GITR and CD103. The prevalence of Tregs increased with during progressive stages in HCC patients. Moreover, the elimination of Treg cells followed by stimulating with NY-ESO-1b peptide significantly improved the anti-tumor cytotoxic T lymphocytes responses in HCC patients compared with stimulating with NY-ESO-1b peptide alone. The immune response efficiency increased from 37.5 to 62.5%. In conclusion, the increase in frequency of Treg cells might play a role in suppression of the immune response against HCC and for the design of immunotherapy the incorporation of the Treg cell depletion strategy will achieve potent anti-tumor immunity with therapeutic impact.

[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.

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.

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.

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.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. 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.