MiR-196a-5p facilitates progression of estrogen-dependent endometrial cancer by regulating FOXO1

Background and Purpose. Estrogen-dependent endometrial cancer mainly occurs in younger pre-menopausal and post-menopausal women and threatens their health. Recently, microRNAs (miRNAs) have been considered as novel targets in endometrial cancer treatment. Therefore, we aimed to explore the effect of miRNA (miR)-196a-5p in estrogen-dependent endometrial cancer. Methods. 17β-estradiol (E2; 2.5, 5, 10 and 20 nM) was used to treat RL95-2, HEC-1B and ECC-1 cells followed by cell viability assessment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The level of miR-196a-5p was measured by reverse transcription-quantitative PCR (RT-qPCR). We then transfected miR-196a-5p mimic/inhibitor and Forkhead box protein O1 (FOXO1) small interfering RNA (siRNA) into E2-treated cells. Apoptotic cells were measured by flow cytometry. Wound healing and Transwell assays were implemented to assess migration and invasion. Bioinformatics and luciferase reporter assays were applied to confirm the interaction between miR-196a-5p and FOXO1. Immunoblotting determined the levels of FOXO1, Bcl-2, Bax, Caspase 3. Results. E2 promoted cell viability and miR-196a-5p expression in RL95-2 and ECC-1 cells. miR-196a-5p mimic enhanced cell viability, migration and invasion but suppressed apoptosis and FOXO1, whilst miR-196a-5p inhibitor blocked these processes. In addition, miR-196a-5p upregulated Bcl-2, but down regulated Bax and Caspase 3 expression, an effect that was reversed by miR-196a-5p inhibitor. We determined that miR-196a-5p targeted FOXO1, and that si-FOXO1 blocked the effects of miR-196a-5p inhibitor on viability, apoptosis, migration and invasion of E2-treated RL95-2 and ECC-1 cells. Conclusions. Our findings suggested potential diagnostic and therapeutic applications for miR-196a-5p and its FOXO1 target in patients suffering from estrogen-dependent endometrial cancer

Identification of a novel conserved HLA-A*0201-restricted epitope from the spike protein of SARS-CoV

<p>Abstract</p> <p>Background</p> <p>The spike (S) protein is a major structural glycoprotein of coronavirus (CoV), the causal agent of severe acute respiratory syndrome (SARS). The S protein is a potent target for SARS-specific cell-mediated immune responses. However, the mechanism CoV pathogenesis in SARS and the role of special CTLs in virus clearance are still largely uncharacterized. Here, we describe a study that leads to the identification of a novel HLA-A*0201-restricted epitope from conserved regions of S protein.</p> <p>Results</p> <p>First, different SARS-CoV sequences were analyzed to predict eight candidate peptides from conserved regions of the S protein based upon HLA-A*0201 binding and proteosomal cleavage. Four of eight candidate peptides were tested by HLA-A*0201 binding assays. Among the four candidate peptides, Sp8 (S_958-966, VLNDILSRL) induced specific CTLs both <it>ex vivo </it>in PBLs of healthy HLA-A2⁺donors and in HLA-A2.1/K^btransgenic mice immunized with a plasmid encoding full-length S protein. The immunized mice released IFN-γ and lysed target cells upon stimulation with Sp8 peptide-pulsed autologous dendritic cells in comparison to other candidates.</p> <p>Conclusion</p> <p>These results suggest that Sp8 is a naturally processed epitope. We propose that Sp8 epitope should help in the characterization of mechanisms of virus control and immunopathology in SARS-CoV infection.</p

A trial of patient-oriented problem-solving system for immunology teaching in China: a comparison with dialectic lectures

BACKGROUND: The most common teaching method used in China is lecturing, but recently, efforts have been widely undertaken to promote the transition from teacher-centered to student-centered education. The patient-oriented problem-solving (POPS) system is an innovative teaching-learning method that permits students to work in small groups to solve clinical problems, promotes self-learning, encourages clinical reasoning and develops long-lasting memory. To our best knowledge, however, POPS has never been applied in teaching immunology in China. The aim of this study was to develop POPS in teaching immunology and assess students’ and teachers’ perception to POPS. METHODS: 321 second-year medical students were divided into two groups: I and II. Group I, comprising 110 students, was taught by POPS, and 16 immunology teachers witnessed the whole teaching process. Group II including the remaining 211 students was taught through traditional lectures. The results of the pre- and post-test of both groups were compared. Group I students and teachers then completed a self-structured feedback questionnaire for analysis before a discussion meeting attended only by the teachers was held. RESULTS: Significant improvement in the mean difference between the pre- and post-test scores of those in Groups I and II was seen, demonstrating the effectiveness of POPS teaching. Most students responded that POPS facilitates self-learning, helps them to understand topics and creates interest, and 88.12% of students favored POPS over simple lectures. Moreover, while they responded that POPS facilitated student learning better than lectures, teachers pointed out that limited teaching resources would make it difficult for wide POPS application in China. CONCLUSIONS: While POPS can break up the monotony of dialectic lectures and serve as a better teaching method, it may not be feasible for the current educational environment in China. The main reason for this is the relative shortage of teaching resources such as space, library facilities and well-trained teachers

Expression of CD39 on FoxP3+ T regulatory cells correlates with progression of HBV infection

BACKGROUND: Although it is known that regulatory T cells (Tregs) can suppress the function of effector T cells, and may contribute to impaired immune response, the precise role of Tregs during the course of hepatitis B virus (HBV) infection remains to be elucidated. A newly identified subset of the CD4(+)Foxp3(+ )Tregs, the CD39(+ )Tregs, has been associated with viral infections and autoimmune diseases. Therefore, we hypothesized that this discrete Treg subset may contribute to the chronic infection of HBV. RESULTS: Initial characterization studies of healthy peripheral CD39(+)FoxP3(+)CD4(+ )T cells revealed that the majority were CD45RA(- )Treg cells. Subsequent analysis of HBV-infected patients (38 asymptomatic HBV carriers (AsCs), 37 chronic active hepatitis B (CAH), 29 HBV-associated acute-on-chronic liver failure (ACLF)) and healthy individuals (25 controls) was conducted to assess association with HBV copy number and the liver injury marker alanine aminotransferase (ALT). A higher percentage of CD39(+ )Tregs was detected within the population of FoxP3(+)CD4(+ )T cells in peripheral blood of AsCs patients. Moreover, the percentage of CD39(+ )Tregs was significantly less in CAH and ACLF patients. The increased proportions of circulating CD39(+ )Tregs were positively correlated with serum viral load, but inversely correlated with serum ALT level. CONCLUSION: These findings not only suggest that CD39(+ )Treg cells may be involved in HBV disease progression but also identify CD39(+ )Tregs as a dynamic immune regulatory cell population that may represent a new target of immunomodulatory therapeutic interventions

Activated IL-23/IL-17 pathway closely correlates with increased Foxp3 expression in livers of chronic hepatitis B patients

<p>Abstract</p> <p>Background</p> <p>Foxp3 protein plays a critical role in mediating the inflammatory response and can inhibit the proinflammatory IL-23/IL-17 pathway. However, the molecular interplay of Foxp3 and the IL-23/IL-17 pathway in patients with chronic hepatitis B (CHB) remains unclear. To this end, we analyzed the expression patterns of Foxp3- and IL-23/IL-17 pathway-related proinflammatory cytokines in 39 patients with acute-on-chronic liver failure, 71 patients with CHB and 32 healthy controls.</p> <p>Results</p> <p>Foxp3 expression was found to be elevated in and mainly expressed by the CD4⁺T cell sub-population of peripheral blood mononuclear cells and liver tissues of patients with hepatitis B. The intrahepatic expression of Foxp3 strongly correlated with the copies of HBV DNA and the concentration of surface antigen, HBsAg. IL-23/IL-17 pathway-related proinflammatory cytokines were also found to be significantly increased in patients' liver tissues, as compared to healthy controls. Moreover, Foxp3 expression was strikingly correlated with the production of these cytokines in liver tissues of CHB patients.</p> <p>Conclusions</p> <p>The closely-correlated increase of Foxp3 and IL-23/IL-17 pathway activity in HBV-infected livers suggests that the proinflammatory IL-23/IL-17 pathway had not been effectively suppressed by the host immune machinery, such as Treg (Foxp3) cells. Constitutive activation of the IL-23/17 pathway, thus, may support the chronic hepatitis B state.</p

Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall’s corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution

Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function

High-level expression and large-scale preparation of soluble HBx antigen from Escherichia coli

The HBx (hepatitis B virus X protein) is a multifunctional regulator of cellular signal transduction and transcription pathways in host-infected cells. Evidence suggests that HBx has a critical role in the pathogenesis of hepatocellular carcinoma. However, the lack of efficient large-scale preparation methods for soluble HBx has hindered studies on the structure and function of HBx. Here, a new pMAL-c2x protein fusion and purification system was used for high-level expression of soluble HBx fusion protein. The high-purity fusion protein was obtained via amylose resin chromatography and Q-Sepharose chromatography. The untagged HBx was efficiently and rapidly purified by Sephadex G-75 chromatography after cleavage by Factor Xa at 23 °C. The purity of active HBx protein was >99% with a very stable secondary structure dominated by α-helix, β-sheet and random structure. The purified HBx protein can be analysed to determine its crystal structure and function and its capabilities as an effective immunogen

Effects of Telbivudine Treatment on the Circulating CD4+ T-Cell Subpopulations in Chronic Hepatitis B Patients

CD4+ T cells serve as master regulators of the adaptive immune response to HBV. However, CD4+ T-cell subsets are heterogeneous, and it remains unknown how the antiviral agents affect the different CD4+ T cell subtypes. To this end, the expressions of signature transcription factors and cytokines of CD4+ T-cell subtypes were examined in hepatitis B patients before and after treatment with telbivudine. Results showed that, upon the rapid HBV copy decrease induced by telbivudine treatment, the frequencies and related cytokines of Th17 and Treg cells were dramatically decreased, while those for Th2 cells were dramatically increased. No obvious changes were observed in Th1 cell frequencies; although, IFN-γ expression was upregulated in response to telbivudine treatment, suggesting another cell source of IFN-γ in CHB patients. Statistical analyses indicated that Th17 and Tr1 (a Treg subtype) cells were the most sensitive subpopulations of the peripheral blood CD4+ T cells to telbivudine treatment over 52 weeks. Thus, Th17 and Tr1 cells may represent a suitable and effective predictor of responsiveness during telbivudine therapy. These findings not only improve our understanding of hepatitis pathogenesis but also can aid in future development of appropriate therapeutic strategies to control viral hepatitis