Bin Ling

Natural killer cells promote immune tolerance by regulating inflammatory TH17 cells at the human maternal–fetal interface

Natural killer (NK) cells accumulate at the maternal–fetal interface in large numbers, but their exact roles in successful pregnancy remain poorly defined. Here, we provide evidence that TH17 cells and local inflammation can occur at the maternal–fetal interface during natural allogenic pregnancies. We found that decidual NK cells promote immune tolerance and successful pregnancy by dampening inflammatory TH17 cells via IFN-γ secreted by the CD56brightCD27+ NK subset. This NK-cell–mediated regulatory response is lost in patients who experience recurrent spontaneous abortions, which results in a prominent TH17 response and extensive local inflammation. This local inflammatory response further affects the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data identify NK cells as key regulatory cells at the maternal–fetal interface by suppressing TH17-mediated local inflammation.

CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells

The identification of developmental stages in natural killer (NK) cells, especially in human NK cells, has lagged for decades. We characterize four novel populations defined by CD11b and CD27, which can represent the distinct stages of human NK cells from different tissues. Nearly all NK cells from peripheral blood are CD11b+ CD27− populations whereas NK cells from cord blood have CD11b+ CD27− and CD11b+ CD27+ populations. Interestingly, we have found large CD11b− CD27− populations of NK cells from deciduas. We also demonstrate that each population could be characterized by unique functional and phenotypic attributes. CD11b− CD27− NK cells display an immature phenotype and potential for differentiation. CD11b− CD27+ and CD11b+ CD27+ NK cells show the best ability to secrete cytokines. CD11b+ CD27− NK cells exhibit high cytolytic function. We demonstrate that human NK cells at different developmental stages have special functions and describe a new model of human NK cell differentiation.

Significance of E-cadherin, β-catenin, and vimentin expression as postoperative prognosis indicators in cervical squamous cell carcinoma ☆,☆☆,★

Although early-stage cervical cancer can be treated by surgery, distant metastases can be life threatening. It has been a challenge to identify reliable biomarkers as indicators of metastasis or poor prognosis. We investigated the prognostic impact of vimentin, E-cadherin, and β-catenin expression measured by immunohistochemistry staining in samples from 135 patients with clinical stage I or II cervical squamous cell cancer and in normal cervical tissues from 55 patients who underwent hysterectomy for reasons other than neoplasia. Down-regulation of E-cadherin and β-catenin was positively related to histologic differentiation (P < .001), metastasis (P < .001), and recurrence (P < .001), whereas up-regulation of vimentin was inversely related to histologic differentiation, metastasis, and recurrence (P < .0001, .020, and .000, respectively). In univariate Cox regression analysis, high expression of E-cadherin or β-catenin was a positive prognostic indicator for overall survival (P < .001 and P < .001, respectively), whereas high expression of vimentin was a negative indicator (P < .001). In multivariate Cox regression analysis, high expression of E-cadherin was a positive prognostic indicator for overall survival (P = .002), whereas high expression of vimentin was a negative indicator (P = .034). The expression of E-cadherin and vimentin was associated with survival, and the 2 proteins were independent prognostic factors in univariate and multivariate analyses. The combination of a decrease of E-cadherin and an increase in vimentin might be a valuable survival indictor in cervical squamous cell cancer.

Establishment, characterization, and successful adaptive therapy against human tumors of NKG cell, a new human NK cell line.

Natural killer (NK) cells play important roles in adoptive cellular immunotherapy against certain human cancers. This study aims to establish a new human NK cell line and to study its role for adoptive cancer immunotherapy. Peripheral blood samples were collected from 54 patients to establish the NK cell line. A new human NK cell line, termed as NKG, was established from a Chinese male patient with rapidly progressive non-Hodgkins lymphoma. NKG cells showed LGL morphology and were phenotypically identified as CD56bright NK cell with CD16-, CD27-, CD3-, αβTCR-, γδTCR-, CD4-, CD8-, CD19-, CD161-, CD45+, CXCR4+, CCR7+, CXCR1-, and CX3CR1-. NKG cells showed high expression of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, CD11c), an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C), and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, IFN-γ). The cytotoxicity of NKG cells against tumor cells was higher than that of the established NK cell lines NK-92, NKL, and YT. NKG cell cytotoxicity depended on the presence of NKG2D and NKp30. When irradiated with 8 Gy, NKG cells were still with high cytotoxicity and activity in vitro and with safety in vivo, but without proliferation. Further, the irradiated NKG cells exhibited strong cytotoxicity against human primary ovarian cancer cells in vitro, and against human ovarian cancer in a mouse xenograft model. The adoptive transfer of NKG cells significantly inhibited the ovarian tumor growth, decreased the mortality rate and prolonged the survival, even in cases of advanced diseases. A number of NKG cells were detected in the ovarian tumor tissues during cell therapy. In use of the new human NK cell line, NKG would a promising cellular candidate for adoptive immunotherapy of human cancer.

Bcl-xL is associated with the anti-apoptotic effect of IL-15 on the survival of CD56dim natural killer cells

Human NK cells can be distinguished into CD56(bright) and CD56(dim) subsets based on cell surface CD56 density. It has been shown that IL-2 and IL-15 have opposing effects on life and death of CD8(+) T cells. However, the roles of IL-2 and IL-15 in regulating these two NK cell subsets remain elusive. In this study, we comparatively analyzed the effects of IL-2 and IL-15 on two NK cell subsets. IL-15 improved the proliferation and activation of CD56(dim) NK cells in long-term cord blood mononuclear cell culture, but IL-2 only maintained the survival of CD56(bright) NK cells. The percentage of CD56(+)Annexin V(+) NK cells cultured with IL-15 was lower than that with IL-2; moreover, most of Annexin V(+) NK cells were primarily in the CD56(dim) NK cells. IL-15 cultured NK cells expressed higher level of Bcl-xL than IL-2 cultured cells. Furthermore, IL-15 more strongly upregulated CD25 expression and better maintained the expression of IL-15Ralpha than IL-2. These results suggest that CD56(dim) NK cells undergo apoptosis when cultured with IL-2, but IL-15 inhibits their apoptosis and Bcl-xL is associated with the anti-apoptotic effect of IL-15. So IL-15 played a crucial role in sustaining long-lasting functions of CD56(dim) NK cells.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbeccos modified Eagles medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

Background Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis. Methodology/Principal Findings The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model. Conclusion/Significance The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.

Induction the cornification of squamous cancerous cells to eliminate tumor cells by promotion cell differentiation and stratum.

Cornification is a kind of apoptosis of squamous epithelial cell in the upper layer of the epithelial tissue. The basal cells away from cell-cycle embark on terminal differentiation, stratum, then die by apoptosis. The critical elements interference from the normal differentiation of squamous cells will lead to cell malignant transformation. We would like to put forward a hypothesis that initiation the cornification of squamous cancerous cells by promotion cell differentiation and stratum can promote squamous carcinoma cells death. Attempts of induction squamous cancerous cells terminal differentiation will add to our understanding of the connection between keratinocytes cornification and the death of squamous-cell carcinoma.

Comparison of laparoscopic peritoneal vaginoplasty and sigmoid colon vaginoplasty performed during radical surgery for primary vaginal carcinoma

BackgroundRadical surgery of primary vaginal carcinoma typically involves partial or complete resection of the vagina, and young patients in particular can experience sexual dysfunction after surgery. Vaginoplasty is mandatory for this population, multiple vaginal reconstructive techniques have been reported. Here we attempted to determine whether the peritoneum is a feasible alternative to the sigmoid colon in vaginoplasty performed during radical surgery.MethodsBetween February 2005 and July 2009, 12 patients underwent radical surgery for Federation of International Gynecology and Obstetrics Stage I primary vaginal carcinoma in the upper one-third of the vagina. To retain a sex life, the patients received vaginoplasty either with the peritoneum (peritoneal group, 5 patients) or with the sigmoid colon (sigmoid group, 7 patients) during radical surgery. Surgeries were performed at the Anhui Provincial Hospital in China. The data between the two groups was retrospectively analyzed.ResultsThe operating time was shorter for the peritoneal group than for the sigmoid group (P < 0.05). There were no significant differences in blood loss as well as in the length or width of the neo-vaginas between the two groups during surgery (P > 0.05). No metastasis or operation-related complications were observed in any of the patients. Six months after surgery, the neo-vaginas of both groups were smooth, soft, and moist. The neo-vaginas in the sigmoid group were similar in size during and 6 months after surgery. The neo-vaginas in the peritoneal group were shorter (although no less wide) 6 months after surgery (P < 0.05); length and width (that admitted two fingers) remained stable thereafter. All patients experienced a satisfactory sex life after surgery. Colposcopy revealed a good vaginal surface covered with squamous epithelium in the neo-vaginas of the peritoneal group, and intestinalization in the neo-vaginas of the sigmoid group. At the 36-month follow-up, all patients were clinically free of disease.ConclusionsLaparoscopic vaginoplasty using the peritoneum compared with using the sigmoid colon is simpler and more feasible for management of Stage I primary vaginal carcinoma. Its benefits include shorter operating time, no bowel disturbance, and production of a hygienic vaginal environment, as well as a potential sex life and oncologic outcome comparable to that of sigmoid colon vaginoplasty.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.