[Show abstract][Hide abstract] ABSTRACT: Jagged-1 signaling has recently been reported to be involved in the Th17 cell differentiation. However, little is known about its mechanisms. Soluble Jagged-1 was used to activate the Jagged-1-Notch signaling to interfere with the IL-6 and TGF-β-induced Th17 cell skewing. Genes relevant to the autoimmunity or inflammation were screened for the first time in this system by qPCR array for the differential expressions. The 18 genes out of 84, including Clec7a, Il12b, Il12rb1, Il12rb2, Csf3, Il15, Il17a, Il17f, Il17rc, Il17rd, Il17re, Il23a, Myd88, Socs1, Stat4, Stat5a, Sykb and Tbx21, were downregulated, but only Cxcl2, Cxcl12 and Mmp3 were upregulated. The expressions of the genes, Rorγt, Il17a, Il17f, Il12rb1 and Il23a, induced by simultaneous IL-6 and TGF-β treatment were significantly suppressed by Jagged-1, followed by the reduction of RORγt, IL-17A, and IL-17F. Consistent with the attenuation of RORγt, and the reduced production and secretion of IL-17A and IL-17F in the cell supernatant and the in situ stained cells, the number of CD4(+)IL-17(+) cells was also diminished. It is concluded that the Jagged-1-Notch signaling can suppress the IL-6 and TGF-β treatment-induced Th17 cell skewing through the attenuation of RORγt and, hence by, the down-regulation of IL-17A, IL-17F, IL-23a, and IL-12rb1.
[Show abstract][Hide abstract] ABSTRACT: Little is known about whether there is a relationshipbetweenPI3K/AKT, ERK1/2 and an inverted CCAAT box binding protein (ICBP90) in biological behaviours of tumour cells. The aim of this study was to determine thisusing Jurkat T cells. Compared to PD98059 (an ERK1/2 signaling inhibitor), DAPT (a Notch signaling inhibitor) or adriamycin (a classical anti-tumour drug), the inhibition of Jurkat T cell growth by LY294002 (a PI3K/Akt signaling inhibitor) was more obvious. LY294002 combined with adriamycin appeared to have a synergistic effect. LY294002 strongly blocked Jurkat T cells at each phase of cell cycle with a decrease of DNA content, superior to adriamycin. Consistent with these changes, the expression of phosphorylated ERK1/2 was markedly decreased in the LY294002-treated Jurkat T cells, leading to the reduction of ICBP90 production, followed by moderate attenuation of TGF-β secretion. The results suggest that deactivation of PI3K/Akt signalling can surpress Jurkat T cell growth through inhibiting cell proliferation and blocking the cell cycle. ICBP90 may mediate the PI3K/AKT-ERK1/2 signalling to regulate leukemia cell growth.
Central European Journal of Biology 08/2014; 9(8). DOI:10.2478/s11535-014-0313-2 · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. It has been found that a quite low dose of anisomycin is sufficient to block proliferation of primary T lymphocytes. The focus of this study is to explore the possibility of anisomycin to treat human acute leukemia Jurkat T cells in vitro. The results indicated that the low dose of anisomycin could significantly inhibit the colony formation of Jurkat T cells and elevate the inhibition rate of Jurkat T cell growth along with its increasing concentrations. Jurkat T cell cycle was blocked into S-phase by anisomycin. Consistent with the increased proportion of sub-G1 phase, anisomycin promoted Jurkat T cell apoptosis. The CD69 and CD25 expression on the surface of Jurkat T cells was also down-regulated prominently along with the enhancing concentrations of anisomycin, followed by the decreased production of IL-4, IL-10, IL-17, TGF-β and IFN-γ, and the down-regulated expression of phosphorylated-ERK1/2. The results suggest that the suppressive effect of anisomycin on Jurkat T cell growth may be related to inhibiting TGF-β production and ERK1/2 activation, arresting the cell cycle at S-phase and promoting the apoptosis of Jurkat T cells.
Central European Journal of Biology 12/2013; 8(12). DOI:10.2478/s11535-013-0235-4 · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ionomycin in conjunction with phorbol-12,13-dibutyrate (PDBu) is conventionally used as a stimulator to activate cells, especially original T cells. But we accidently found it had an entirely opposite action on malignant tumor cells derived from T cells. Thus, influence of ionomycin on human leukemia Jurkat T cell behaviors and its preliminary mechanistic process were explored in the presence of PDBu. Ionomycin could remarkably inhibit colony formation of the cells, and inhibitory rate of the cell proliferation was increased with ionomycin treatment in a dose- or time-related relationship, following the reduction of ERK1/2 and phosphorylated-ERK1/2 levels. However, a high dose of ionomycin might moderately repress mid-stage activation of the cells. It also blocked the cell entry at S-phase and G2/M-phase with the attenuation of transforming growth factor-β (TGF-β) level in the cells, and promoted the cell apoptosis following the augment of caspase-3 and cleaved caspase-3 in the cells. The dramatic elevation of [Ca2(+)]i and intracellular pH (pHi) was simultaneously followed by the above alteration of the cell behaviors. These results indicate that ionomycin may strongly inhibit human acute T lymphocyte leukemia progress in the presence of PDBu through the inhibition of ERK1/2 signaling, the activation of caspase-3 and the attenuation of TGF-β mediated by the [Ca2(+)]i and pHi enhancement, providing a novel insight into function and potential application of both ionomycin and PDBu.
Annals of Hematology 11/2013; 93(5). DOI:10.1007/s00277-013-1955-2 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of Notch by Jagged-1 may plays a pivotal role in maturation of dendritic cells (DCs), but the mechanism has not been completely defined. In the present study, Hes-1 (Hairy/enhancer-of-split)-targeting siRNA was used to confirm a role of Jagged-1-Notch signaling pathway activation in maturation of murine bone marrow-derived DCs and to search for a target that plays a critical role. The results showed that compared with the control, lipopolysaccharide or Zymosan A groups, Jagged-1 (a soluble Jagged 1/Fc chimera protein) effectively increased expression of Hes-1 and Deltex-1 mRNA, which could be reversed by DAPT (2, 4-diamino-5-phenylthiazole), a specific inhibitor of the Notch signaling pathway. Hes-1-targeting siRNA could successfully down-regulate the endogenous Hes-1 expression in the DCs. Concurrently, a significant down-regulation of CD40, CD80, CD86 and MHC-II expressions on the surface of the DCs was found with the reduction of IL-12 yielded by the DCs. Our results demonstrate that Hes-1-targeting siRNA can inhibit the maturation of the DCs induced by Jagged-1, indicating Hes-1 may be an important target of Notch signaling mediating the maturation of DCs.
Central European Journal of Biology 11/2013; 8(11). DOI:10.2478/s11535-013-0231-8 · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anisomycin eminently inhibits cell proliferation in vitro. The aim of this study was to explore the potential of anisomycin to treat tumors in vivo and its mechanism(s) of action. The results showed that peritumoral administration of anisomycin significantly suppressed Ehrlich ascites carcinoma (EAC) growth resulting in the survival of approximately 60% of the mice 90 days after EAC inoculation. Enhancement of infiltrating lymphocytes was noted in the tumor tissue, which was dramatically superior to adriamycin. The growth inhibitory rate of EAC cells was enhanced with increasing concentrations of anisomycin, following an enhanced apoptotic rate. The total apoptotic rate induced by 160 ng/ml of anisomycin was higher when compared to that induced by 500 ng/ml of adriamycin. DNA breakage and nanostructure changes were also noted in the EAC cells. The levels of caspase-3 mRNA, caspase-3 and cleaved-caspase-3 proteins in the anisomycin‑treated EAC cells were augmented in a dose- and time-dependent manner, following the activation of caspase-8 and caspase-9, which finally triggered PARP cleavage. The cleaved-caspase-3, cleaved-caspase-8 and cleaved-caspase-9 proteins were mainly localized in the nuclei of the cells. These results indicate that anisomycin efficaciously represses in vitro and in vivo growth of EAC cells through caspase signaling, significantly superior to the effects of adriamycin. This suggests the potential of anisomycin for the treatment of breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Notch signaling plays an important role in differentiation of T cells. However, little is known as to action of it in differentiation of Th17 cell subset. In this study, a soluble Jagged-1/Fc chimera protein (Jagged-1) was directly used to activate Jagged-1Notch signaling, while Hes-1-targeting siRNA was used to knock down Hes-1 gene to investigate effect of Jagged-1Hes-1 signaling on the differentiation of CD4+ T cells into Th17 cells. The results showed that Jagged-1 could promote the expression of Hes-1 and Deltex-1 mRNAs and the expression of NICD, Hes-1 and Deltex-1 proteins, which might be significantly blocked by DAPT, a specific inhibitor of Notch signaling. Jagged-1Hes-1 signaling resulted in the markedly decreased in situ expression of RORgammat in the CD4+ T cells induced by IL-6 plus TGF-ß. Flow cytometric analysis showed the reduction of IL-17 production in CD4+ T cells by Jagged-1, but the enhancement of it by Hes-1-targeting siRNA. The level of IL-10 produced by the treated cells was also enhanced, whereas the expression of IL-17 was prominently attenuated, which could be offset by anti-Jagged-1 antibody or DAPT. The results indicate that Jagged-1Hes-1 signaling can suppress the skewing of CD4+ T cells toward Th17 cells via RORgammat, for which Hes-1 may be crucial.
Journal of biological regulators and homeostatic agents 03/2013; 27(1):79-93. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Recent studies have shown that anisomycin significantly inhibits mammalian cell proliferation, but its mechanism remains unclear. In this study, Jurkat T cells were used to first explore a relationship between effect of anisomycin on them and alteration of cell cycle-regulating proteins.
Cell colony formation, CCK-8 assay, flow cytometry, RT-PCR and western blot were employed to evaluate correlation of ten cell cycle-regulating proteins with suppression of the cell proliferation and arrest of the cell cycle by anisomycin.
Our data showed that anisomycin inhibited the colony-formation and proliferation of Jurkat T cells in a dose-dependent manner, and arrested the cells into S and G2/M phases with the production of sub-diploid cells. The levels of P21, P-P27 and P53/P-P53 reached their peaks 4 h after anisomycin treatment, presenting a positive correlation with anisomycin concentration, and P16, P-P21, P27, P57, P73/P-P73 and P-Rb changed little with the prolonged exposure time or increased concentrations of anisomycin. But the level of Rb protein was increased at 24 h after the treatment of anisomycin. The expression of an inverted CCAAT box binding protein (ICBP90) in Jurkat T cells came to decrease 12 h after the treatment of anisomycin, presenting a negative correlation with anisomycin concentration. Subsequently, the expression of P-CDK2 was also decreased at 24 h, presenting an obviously negative correlation, whereas P-CDK1 showed no differences among the differently treated Jurkat T cells. Furthermore, the level of P21 and P53 mRNA was increased with the enhanced concentrations of anisomycin.
The results indicate that anisomycin may activate the P53/P21/P27 signaling to decrease the expression of ICBP90, inhibit expression of P-CDK2 to block the cells into S and G2/M phases, and finally result in proliferation inhibition of Jurkat T cells.
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptor (TLR)-TLR cross talk is thought to be important in TLR signaling. Herein, we investigated the effect of specific TLR3 and TLR7 agonists, poly (I:C) and R837, individually and in combination, on uterine immune cell function and their subsequent effects on pregnancy outcome. Allogeneic pregnancies in the non-obese diabetic (NOD) mousexC57BL/6 and wild-type BALB/cxC57BL/6 model were used. An additive increase in embryo resorption was observed after induction with both poly (I:C) and R837, and was associated with elevated numbers of both TNF-alpha- and IFN-gamma-producing CD45(+) cells in the uterus. Further examination showed that while cytokine expression was detected in both CD3(+) cells and CD49b(+) cells in BALB/c mice, NOD mouse cells behaved differently. In NOD mice, elevated cytokine expression was attributed to CD3(+) T cells, with no response detected in the CD49b(+) NK cells. The additive effect of combined agonists was partially inhibited by the Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) inhibitor SP600125 and almost completely abrogated by the extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. These results suggest that increased TLR3 and TLR7 signals are transmitted via Th1-type T cells, rather than NK cells, in NOD mice. Furthermore, the ERK MAPK pathway may be critical in TLR3 and TLR7 signaling.
[Show abstract][Hide abstract] ABSTRACT: Both regulatory T cells and regulatory natural killer (NK) cells may play essential roles in the maintenance of pregnancy. In this study, we show that a significantly high percentage of spontaneous embryo loss was observed in both allogeneic and syngeneic pregnant non-obese diabetic (NOD) mice. The percentage of embryo loss in allogeneic pregnant mice was further increased by the administration of anti-asialo ganglio-N-tetraosylceramide to deplete NK cells, but was decreased by the adoptive transfer of ITGA2(+)ISG20(+) (CD49b(+) CD25(+)) NK cells from normal mice. No such trend was observed in syngeneic pregnant NOD mice. The pattern of CXCR4 (specific receptor for CXCL12) expression on NK cells was analyzed and NK-cell migration was confirmed by in vitro and in vivo migratory assays. Since CXCL12 production by murine trophoblast cells was confirmed previously, our findings suggest that the recruitment of peripheral CXCR4-expressing ITGA2(+)ISG20(+) NK cells into pregnant uteri may be important in the regulation of feto-maternal tolerance.
[Show abstract][Hide abstract] ABSTRACT: To investigate the role that CD25(+) natural killer (NK) cells may play in the establishment of pregnancy, the effect of adoptive CD25(+) NK cell transfer on pregnancy outcome in subfertile non-obese diabetic (NOD) mice was investigated. Phenotypic analysis of NK cells was performed by flow cytometry before and after the transfer. The proportion of subfertile NOD female mice that failed to become pregnant when co-caged with C57Bl/6 males for 16 weeks was significantly higher in female NOD mice than in normal female BALB/c controls (53.1% versus 15.1%; P < 0.01). The subfertile NOD mice were divided into three groups receiving CD25(+) NK cells (group 1), CD25(-) cells (group 2) or RPMI 1640 medium only (group 3). Group 1 had significantly more pregnancies than those receiving CD25(-) NK cells (77.8% versus 11.1%; P < 0.01) and controls injected with RPMI 1640 medium (0.0%; P < 0.01). Improved fertility was concomitant with an increase in placental CD49b(+) NK cells expressing Foxp3. Foxp3 expression was confirmed in the CD25(+) NK cells before the transfer. These results indicate that subfertility in NOD mice may be partially attributed to the insufficiency of CD25(+) and Foxp3(+) NK cells recruited into the pregnant uterus.
[Show abstract][Hide abstract] ABSTRACT: We confirmed previously the existence of thymic stromal lymphopoietin (TSLP)-positive cells in murine placenta by flow cytometry. To compare the characteristics of Toll-like receptor 3 (TLR3)- and TSLP-mediated placental dendritic cell (DC) activation, pregnant BALB/c mouse mated with C57BL/6 male were used as a model of allogenic gestation. Placental CD11c(+) DCs were potently activated by the TLR3-agonist polyinosinic polycytidylic acid [poly (I:C)], subsequently causing increased expression of co-stimulatory molecules. Accordingly, increased intracellular production of interleukin (IL)-12 and interferon (IFN)-gamma, but not IL-4 or IL-10, were detected after stimulation by poly (I:C). In the case of TSLP-stimulation, although increased expression of co-stimulatory molecules was also detected, there was no substantial increase of intracellular production of IL-12, IFN-gamma, IL-4 or IL-10. In contrast, the expression of the Th2 cell-attracting chemokine, the thymus and activation-regulated chemokine (TARC) or CCL17, was significantly boosted in response to TSLP induction, whereas no significant increase of CCL17 was observed when triggering TLR3 with its specific agonist poly (I:C). The data were further supported by a CD4(+)IL-10(+) cell migratory assay. These results suggest that TSLP-TSLP receptor interaction may result in a Th2-type microenvironment at the feto-maternal interface by inducing the production of Th2 cell-attracting chemokine and modulating the immigration of Th2-type cells.
[Show abstract][Hide abstract] ABSTRACT: There is still a lack of a high potent and low toxic immunosuppressive drug. We accidentally found that a quite low dose of anisomycin was sufficient to block proliferation of T cells. In this study, carboxy-fluorescein diacetate-succinimidyl ester staining showed that over 10.0 ng/mL of anisomycin markedly inhibited the proliferation of T cells induced by ConA. Propidium iodide staining revealed that anisomycin led to G0/G1 arrest and blocked S phase entry stimulated by ConA or phorbol 12, 13-dibutyrate plus ionomycin. Anisomycin down-regulated remarkably the CD69 and CD25 expression on the surface of T cells. The response of T cells was repressed by treatment of anisomycin, which was partly restored by adding exogenous interleukin-2, and there was no difference between anisomycin and dexamethasone, although the used dose of the latter was 100-fold of the former. The inhibition of cytotoxicity of T cells against 7919 cells by anisomycin was observed without the direct cytotoxicity to T cells or 7919 cells. The level of transforming growth factor-beta1 fell by <80.0 ng/mL in vitro and 30.0 mg/kg of anisomycin in vivo and enhanced by more than the doses. The treatment of anisomycin prolonged the survival of the transplanted skin and depressed the delayed type hypersensitivity development and the T-cell response in the skin-transplanted mice. Moreover, the effect of its restraining allograft rejection might be superior to cyclosporine A, with relatively slight toxic signs. These results indicate anisomycin significantly inhibits the behaviors of T cells and the transplantation rejection, providing important evidence for anisomycin as a novel immunosuppressant.
[Show abstract][Hide abstract] ABSTRACT: To characterize uterine natural killer (uNK) cells in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice and investigate the potential role of these cells in pregnancy tolerance.
An animal model-based study.
Academic research center in a university.
Syngeneic pregnant NOD/SCID mice were compared with non-immunodeficient BALB/c mice.
Induction of Toll-like receptor (TLR) agonists.
Flow cytometric analysis was performed to detect the percentage of cell subsets, and standard (51)Cr release assay was performed to determine cytotoxicity.
The dominant subset of uNK cells in NOD/SCID mice is DX5 (CD49b)(+), asialo ganglio-N-tetraosylceramide(+), CD25(+), CD122(+), Thy-1 (CD90)(hi), c-kit (CD117)(hi), and interleukin-10(+). In addition, the percentage of interferon-gamma(+) subset was slightly increased in response to selected TLR agonists in the NOD/SCID mice, whereas the corresponding percentage in BALB/c mice could be increased dramatically. Such an effect could be abrogated by inhibitors, including LY294002, SP600125, and PD98059. The significant increase of interferon-gamma(+) NK cell percentage in BALB/c mice was concomitant with the increase of the embryo resorption rate. In contrast, the resorption rate in NOD/SCID mice was not significantly increased upon the induction of polyinosinic polycytidylic acid or lipopolysaccharide. As expected, the NK cells from NOD/SCID mice display a detectable but lower cytotoxicity than BALB/c, as determined by standard (51)Cr release assay. In addition, the uNK cells from NOD/SCID mice also display a hyposensitivity to lipopolysaccharide-induced production of inducible nitric oxide synthase.
A considerable percentage of immature NK cells were detected at the fetomaternal interface in NOD/SCID mice. These cells were hyposensitive to the stimulation of selected TLR agonists. Such a status seemed to be beneficial for the maintenance of pregnancy.
Fertility and sterility 05/2008; 91(6):2676-86. DOI:10.1016/j.fertnstert.2007.08.087 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thymic stromal lymphopoietin (TSLP)-TSLP receptor (TSLP-R) interactions activate CD11c(+) dendritic cells (DCs) and increase epithelial cell Th2-type cytokine production. We detected intracellular TSLP expression on CK7(+) trophoblast cells and TSLP-R expression on placental DCs from pregnant BALB/cxC57BL/6 and NOD/SCIDxC57BL/6 mice on gestational day 12.5. Murine recombinant TSLP activated DCs from BALB/c mice, with increased CD80 and CD83 expressions; TSLP-activated DCs induced IL-10-producing NK cell expansion. This was abrogated by anti-TSLP Ab or by culturing CD49b(+) NK cells alone. No TSLP-DC-induced IL-10(+)CD49b(+) cell expansion occurred when DCs and CD49b(+) cells were cultured separately. Although TSLP-induced DC activation occurred in NOD/SCID mice, the IL-10(+) NK cell percentage was unchanged. CK7(+) trophoblast cells may activate placental DCs via a TSLP-TSLP-R interaction and induce DC-dependent placental NK cell IL-10 production. TSLP-DC and NK cell contact appears necessary for IL-10(+)CD49b(+) cell expansion. Placental NK cells from NOD/SCIDxC57BL/6 mice appear less sensitive to TSLP-DC induction.
[Show abstract][Hide abstract] ABSTRACT: To investigate the potential role that toll-like receptor 3 (TLR3) may play in a murine model of polyinosinic-polycytidylic acid (polyIC)-induced embryo resorption.
An animal model-based study on induced embryo resorption.
University animal laboratory.
Pathogen-free animals housed under barrier conditions and monitored for health status. Pregnant BALB/c mouse mated by C57BL/6 male was used as a model of allogeneic gestation.
The administration of polyIC was performed to establish a murine model of induced embryo resorption, with or without TLR3 blocking by multiple injection of mAb against this receptor.
Flow cytometric analysis was performed to detect the percentage of CD45(+)DX5+ and DX5(+)CD69+ cell subsets, with the DX5 antigen used as a common natural killer (NK) cell marker, and CD69 as a marker for activated NK cells, respectively.
In the allogeneic mating model, BALB/cxC57BL/6, both the CD45(+)DX5+ and DX5(+)CD69+ cell percentages were significantly elevated upon polyIC stimulation at the absence of anti-TLR3 administration but were kept unchanged if the female mice were pretreated with anti-TLR3 monoclonal antibody. Accordingly, the resorption rate of embryos was boosted by polyIC administration, but this effect could be abrogated by pretreatment of anti-TLR3 mAb.
The engagement of polyIC with TLR3 may be critical to the activation of NK cells infiltrated at the feto-maternal interface, subsequently resulting in an increase in embryo resorption.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to outline the fertility features of non-obese diabetic (NOD)/LtSz-scid/scid (NOD/SCID [severe combined immunodeficiency] for short) mice, and to evaluate the effects of NK cell subsets on the pregnancy outcomes of the syngeneic NOD/SCID x NOD/SCID mating combination. Firstly, lymphocyte phenotyping was performed with flow cytometry to confirm the multiple immunodeficits in NOD/SCID mice. Fertility features were assessed in NOD/SCID x NOD/SCID mice and compared with non-immunodeficiency control BALB/c x BALB/c mice. Although the presence of NK cell deficit is apparent in NOD/SCID mice, a certain level of remnant NK activity could be observed in these mice. The remnant NK cell activity was stimulated with polyinosinic-polycytidylic acid (polyIC), or inhibited with anti-asialo GM1 (ASGM1) anti-serum, respectively. The effects of these factors on pregnancy outcomes were evaluated after administration. Roughly normal fertility could be observed in NOD/SCID x NOD/SCID mice. However, a slightly larger sized litter was observed in polyIC-treated NOD/SCID x NOD/SCID mice compared with control NOD/SCID mice. In contrast, embryo resorption was boosted after ASGM1 injection, and correlated subsequently with a smaller litter size. It indicates that the remnant NK cell activity in NOD/SCID mice may be beneficial to feto-maternal tolerance during pregnancy.
[Show abstract][Hide abstract] ABSTRACT: Stem cells in healthy epidermis have the capacity to produce structures, such as interfollicular epidermis, hair follicle, and sebaceous glands. Embryonic stem (ES) cells can be induced to differentiate into epidermal stem cells in vitro. These ESCs (epidermal or epidermoid stem cells derived from ES cells) have the potential to be an ideal replacement of those stem cells destroyed in severe injury, such as in deep and extensive burn. The aim of this study was to follow the fate of murine ESCs which were seeded in a syngeneic dermal equivalent and implanted into syngeneic recipient mice subcutaneously.
ES cells were induced in vitro to differentiate into ESCs. Stained with a fluorescent dye Hoechst 33342, these ESCs were seeded into a fibroblast-collagen-gelatin sponge complex, functioning as a dermal equivalent model, and then implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells.
These ESCs were clearly visible in the implant by fluorescent microscopy 3 weeks or longer after implantation. These cells remained viable, differentiating into hair follicle-like structures, glandular structures, and gave rise to additional structures resembling native dermis. A number of markers were expressed in the differentiated structures, including CD29 (integrin beta1 subunit) and cytokeratin 18 (CK18). No apparent rejection or severe side effects were observed at least during the 10 weeks following implantation.
Now that ESCs can survive in vivo in this dermal equivalent model and differentiate into hair follicle-like structures as well as glandular structures, it is feasible to use these cells as seed cells in studies to fabricate dermal equivalents that have the potential to develop dermal appendages.
[Show abstract][Hide abstract] ABSTRACT: Cytokeratin 7 (CK7) is currently regarded as the best marker for trophoblast cells, while CD200 (OX-2), known as 'tolerance signal', plays an important role in normal pregnancy. In this study, the status of CD200 expression was investigated in BALB/c x C57BL/6 and BALB/c x BALB/c mating combinations designed as allogeneic and syngeneic murine models of induced embryo resorption, in which the resorption rate was boosted by an i.p. injection of poly (I:C), a synthetic double-stranded RNA. The percentage of CD200+ cells in the CK7+ cell population (CD200+ CK7+ percentage) and the absolute number of these cells were determined with flow cytometry, using trophoblast cells collected at day 8.5 and day 13.5 of gestation. The potential effect of poly (I:C) on CD200 expression was also evaluated by detecting the CD200+ CK7+ percentage in trophoblast cells incubated in the presence or absence of poly (I:C), in vitro. The distribution pattern of CD200+ cells at the feto-maternal interface was evaluated by immunocytochemical examination. When 10(4) cells were analyzed at day 8.5 of gestation in each case, no significant difference was observed between the poly (I:C)-treated group and the control PBS group either in the CD200+ CK7+ percentage or in the absolute number of these cells. Similar results were observed both in BALB/c x C57BL/6 mice and in BALB/c x BALB/c mice. However, the CD200+ CK7+ percentage was significantly decreased in the poly (I:C)-treated group when evaluated at day 13.5 of gestation. Accordingly, a dramatically elevated rate of embryo resorption was observed at this time point of pregnancy after the administration of poly (I:C). In addition, the CD200+ CK7+ percentage was significantly lower in trophoblast cells incubated with poly (I:C) at a certain concentration, in vitro, while histocytochemical examination showed the CD200+ cells mainly scattered in placental tissue adjacent to the interface of the placenta and uterus. This indicates that sufficient expression of the CD200 molecule on CK7+ cells at the feto-maternal interface may be necessary for the maintenance of embryos during pregnancy in this rodent model, while poly (I:C) administration may increase embryo resorption, at least partially via direct inhibition of CD200 expression on CK7+ cells.
[Show abstract][Hide abstract] ABSTRACT: The present study aims to address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (pLNs) is valuable in assessment of the status of local immunity at the murine feto-maternal interface. CBA/J x DBA/2 mice, virgin CBA/J mice, and CBA/J x BALB/c mice were used as an abortion-prone model (group A), nonpregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cells in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined by means of flow cytometry (FCM), using mononuclear cells isolated from pLNs collected 5.5, 9.5, and 13.5 days post-coitum (dpc), respectively, and mononuclear cells isolated from placentas 13.5 dpc. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19, and DX5 as specific markers for murine T-cells, B-cells, and NK cells, respectively. Both resorption rate and absolute number of resorptions were significantly higher in group A (29.3%, 1.8+/-1.0) than in group F (4.8%, 0.3+/-0.5, P<0.001, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in pLNs collected 13.5 dpc were significantly higher in group A than in group F (27.5+/-14.0% versus 12.3+/-7.1%, and 1362+/-687 versus 615+/-353, P=0.001, respectively). The CD45+CD86+ percentage was around 7.5% in nonpregnant CBA/J mice, similar to the 10.6% in CBA/JxDBA/2 mice 5.5 dpc, but had increased dramatically, to 23.9%, by 9.5 dpc (P<0.001 versus nonpregnant mice and P=0.002 versus CBA/JxDBA/2 mice 5.5 dpc), and remained at a higher level (27.5%) until 13.5 dpc. However, this trend was not observed in group F during pregnancy. The increased CD45+CD86+ percentage at day 9.5 of gestation, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.