Expression Of LIF Research Articles

Comparison and immunobiological characterization of retinoic acid inducible gene-I-like receptor expression in mesenchymal stromal cells

Due to their immunomodulatory and regenerative properties, Mesenchymal stromal cells (MSC) have generated major interests in several clinical settings including transplantation and inflammatory diseases. MSC functions can be influenced by their tissue origin. Their microenvironment strongly affects their biology notably through TLR sensing. In this study, we show that MSC isolated from four different sources express another type of cytosolic pathogen recognition receptors known as retinoic acid inducible gene-I (RIG-I)-like receptors (RLR). RLR activation in MSC induces the production of Type I IFN (IFN-β) and Type III IFN (IFN-λ1). The highest producers are adipose tissue(AT)-MSC. We further show that Interferon production is induced through TBK1/IKK-ε signaling and IRF7 phosphorylation. Depending on MSC source, the knockdown of TLR3 and/or RIG-I decreases the MSC response to RLR ligand poly(I:C)/Lyovec. Among the different MSC types, AT-MSCs display the highest sensitivity to viral stimuli as shown by the alteration of their viability after prolonged stimulation. Our work indicates that this could be linked to an increase of pro-apoptotic Noxa expression. Finally, the expression of IDO1 and LIF upon RLR activation indicate the increase of MSC immunomodulatory potential, especially in AT-MSCs. Altogether, these data should be considered when designing MSC-based therapy in clinical settings where inflammation or infection are present.

TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs.

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9ON ) or the control LacZ (LacZON ). When engrafted into mice and treated with tetracycline, Tlr9ON , but not LacZON , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL-6-type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9-expressing prostate cancer cells and PMN-MDSCs. Antibody-mediated LIF neutralization reduced the percentage of tumor-infiltrating PMN-MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN-MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF-mediated expansion and activation of PMN-MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide-based inhibitors, such as CpG-STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.

Nasal airway epithelial cell IL-6 and FKBP51 gene expression and steroid sensitivity in asthmatic children.

BackgroundMany asthmatic patients exhibit uncontrolled asthma despite high-dose inhaled corticosteroids (ICS). Airway epithelial cells (AEC) have distinct activation profiles that can influence ICS response.ObjectivesA pilot study to identify gene expression markers of AEC dysfunction and markers of corticosteroid sensitivity in asthmatic and non-asthmatic control children, for comparison with published reports in adults.MethodsAEC were obtained by nasal brushings and primary submerged cultures, and incubated in control conditions or in the presence of 10 ng/ml TNFalpha, 10-8M dexamethasone, or both. RT-PCR-based expression of FKBP51 (a steroid hormone receptor signalling regulator), NF-kB, IL-6, LIF (an IL-6 family neurotrophic cytokine), serpinB2 (which inhibits plasminogen activation and promotes fibrin deposition) and porin (a marker of mitochondrial mass) were determined.Results6 patients without asthma (median age 11yr; min-max: 7–13), 8 with controlled asthma (11yr, 7–13; median daily fluticasone dose = 100 μg), and 4 with uncontrolled asthma (12yr, 7–14; 1000 μg fluticasone daily) were included. Baseline expression of LIF mRNA was significantly increased in uncontrolled vs controlled asthmatic children. TNFalpha significantly increased LIF expression in uncontrolled asthma. A similar trend was observed regarding IL-6. Dexamethasone significantly upregulated FKBP51 expression in all groups but the response was blunted in asthmatic children. No significant upregulation was identified regarding NF-kB, serpinB2 and porin.ConclusionLIF and FKBP51 expression in epithelial cells were the most interesting markers of AEC dysfunction/response to corticosteroid treatment.

The P2X7 receptor links mechanical strain to cytokine IL-6 up-regulation and release in neurons and astrocytes.

Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) invivo, and stretch or swelling invitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60mmHg for 4h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response.

Прогнозування невдалих спроб застосування допоміжних репродуктивних технологій

The objective: development of algorithm of forecasting of unsuccessful attempts of auxiliary genesial technologies on the basis of the mathematical analysis of the main reasons and risk factors. Patients and methods. 97 married couples which passed sterility treatment on the basis of clinic of auxiliary genesial technologies were surveyed. The complex of the conducted researches included clinical, endocrinologic, immunohistochemical, ehografical and statistical methods. Results. For forecasting of unsuccessful attempts of auxiliary genesial technologies it is necessary to use the constructed mathematical model which includes 4 most significant changes (as significance value depression): optical density of an expression of LIF in superficial and glandular epithelium the endometrium, received in the period of assumed «implantation windows» in cycle before carrying out an extracorporal fertilization, the maintenance of VEGF in cervical slime in day of transvaginal puncture, S/D and IR of spiral arteries in day of introduction of triger of an ovulation. Conclusion. Use of offered algorithm allows to increase forecasting effectiveness of unsuccessful attempts of auxiliary genesial technologies. Key words: auxiliary genesial technologies, unsuccessful attempts, forecasting.

Abstract IA14: Targeting Ras

Abstract We are exploring multiple ways of targeting Ras proteins and Ras-driven cancers. We have identified compounds that covalently bind to cysteine-185 on K-Ras 4B, and to the G-domain of K-Ras 4A and 4B. We are also screening for compounds that bind directly to K-Ras using a new technique referred to as Second Harmonic Generation and are developing compounds that promote phosphorylation of K-Ras4B on serine-181. In addition, we are attempting to solve novel structures of oncogenic Ras proteins on their own, or complexed with effectors, regulators and chaperones, in an attempt to identify new targeting opportunities. For example, we have solved the structure of fully processed K-Ras 4B bound to PDE-delta. This co-structure suggests several new approaches to targeting K-Ras processing. In addition to targeting K-Ras directly, we are validating downstream dependencies that are specific for K-Ras cancers, including the cytokine LIF, which is expressed at high levels in K-Ras transformed cells and plays a major role in stem-like properties of K-Ras cancers. Suppressing LIF expression using shRNA, CRISPR or monoclonal antibodies has profound effects on K-Ras tumor initiation and maintenance, and suggests new therapeutic opportunities. We are also analyzing signaling pathways that differ between Ras mutants and between Ras isoforms. These results, and their opportunities for intervention, will be discussed. Citation Format: Frank McCormick. Targeting Ras. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA14.

Immunohistochemical criteria for endometrial receptivity in I/II stage endometriosis IVF-treated patients

sBackground: Implantation failure of in vitro fertilization (IVF) cycles is recognized as one of key problems in contemporary reproductive medicine. Implantation itself is a multifactorial process and one can hardly expect to find a single criterion for the endometrium receptivity. Endometrium biopsy still remains the most applicable technique to diagnose abnormalities causing decrease or complete loss of endometrial receptivity.Materials and methods: We have studied 95 endometrial biopsy samples from 45 patient with I/II stage endometriosis and 40 controls from October 2014 to December 2015. Immunohistochemical analysis of key biological molecules participating in implant window formation (LIF, ER, PR, integrin, TGF-β1 and VEGF) was done to assess their predicting value for endometrial receptivity troubles.Results: The discriminant analysis demonstrated that highest information capacity was characteristic for LIF expression percent area, integrin αVβ3 both percent area and optic density in endometrial stroma and glands and finally TGFβ1 and VEGF-А percent area expression in endometrial stroma. The model test done on a checking group showed 89.1% correct discrimination. Cross-checking in a teaching group showed a bit lower but still high correct answer percentage (88.8%). A decision-making classification tree was worked out.Conclusion: The produced model is sufficient for predicting IVF treatment failure and allows producing reasonable treatment tactics as well as encourages IVF treatment effectiveness improvement in patients with endometriosis.

336 - Molecular mechanisms involved in the immunomodulation induced by LIF in cancer

LIF is a pleiotropic cytokine that can act as an immunomodulatory factor in different biological processes, such as embryo implantation, organ transplantation tolerance and multiple sclerosis. Here, we have observed that in tumors expressing LIF, its blockade inhibits tumor growth, and that the anti-tumor effect of LIF neutralization is mediated by the polarization of tumor-associated myeloid cells (TAMCs). LIF sustains the expression of CCL22, MRC1 and CD163 in human healthy monocytes and in TAMCs from mouse syngeneic (non-small cell lung cancer, ovarian cancer and colorectal cancer) models, patient-derived xenograft models and organotypic cultures from glioblastoma. We show that LIF blockade down-regulates CCL22 secretion by TAMCs preventing regulatory T cell and inducing effector T and NK cell tumor infiltration, leading to an increase in tumor cell apoptosis and an anti-tumor response. A positive correlation between M2-like markers and LIF expression has been observed in glioblastoma patients, and high levels of these factors confer poor prognosis. Moreover, we have studied one of the multiple mechanisms through which LIF could modulate the inhibition of effector T and NK cells: the regulation of PD-1/PDL1 axis. We have seen that after anti-LIF treatment of tumor-bearing mice, the expression of PDL1 decreases in tumor cells and in TAMCs. Furthermore, a positive correlation between PDL1 and LIF expression has been observed in glioblastoma patients, and both proteins also correlate with CD44 expression, a marker for cancer-initiating cell (CIC) population. High levels of these three factors confer poor prognosis. Thus, we have found that LIF acts as an immune-suppressor in cancer recapitulating its normal function in other biological processes. LIF is a bridge between the innate and adaptive immune responses through the induction of CCL22 in TAMCs and subsequent regulation of regulatory and effector T and NK cells. Our results identify LIF as a promising immuno-oncology therapeutic target and establish the translational potential of anti-LIF agents.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

Impaired endometrial receptivity in primary infertility in women with undifferentiated connective tissue dysplasia and hereditary thrombophilia

to investigate endometrial morphological and immunophenotypic features in patients with primary infertility in the presence of uCTD and HT. The pipelle endometrial biopsy specimens taken in the implantation window were examined in 81 patients, including 13 women with a clinical diagnosis of uCTD, 40 with HT, 19 with uCTD concurrent with HT, and in a control group of 9 heathy surrogate mothers. Morphological, immunohistochemical, and morphometric examinations were done to study the paraffin-embedded endometrial biopsy sections stained with hematoxylin and eosin, pikrofuksin by van Gieson, and with toluidine blue. Immunohistochemical tests were carried out using primary antibodies against ER, PgR, LIF, PAI-1, VEGF, Collagen I, Collagen III, fibronectin, laminin, MMP-2, and MMP-9. The uCTD, HT, and uCTD + HT groups were found to have signs of decreased endometrial receptivity as dramatically lower counts of mature pinopodes, slower endometrial maturation, reduced expression of the receptivity marker LIF, and deviations of the stromal progesterone-estrogen index from the normal value. Sclerotic foci with type III collagen accumulation were detected in the endometrial stroma. uCTD and HT and especially their concurrence are commonly a concomitant disease and risk factors for infertility in women due to impaired endometrial receptivity. In uCTD, connective tissue remodeling processes are substantially retarded, which ultimately leads to increased processes of endometrial stromal sclerosis, reduced endometrial receptivity, and infertility. The most pronounced morphological and immunophenotypical changes have been ascertained to develop in the uCTD + NT group. The findings may be used to predict and devise new infertility treatments in patients with uCTD + NT.

Expression of Endometrial Receptivity Genes Increase After Myomectomy of Intramural Leiomyomas not Distorting the Endometrial Cavity.

This study was designed to investigate whether endometrial receptivity genes are altered in infertile patients with intramural leiomyomas (IM) not distorting the endometrial cavity undergoing myomectomy. We measured endometrial HOXA-10, HOXA-11, LIF, ITGB3, and ITGAV messenger RNA (mRNA) expressions levels before and after myomectomy/metroplasty during mid-luteal phase in participants with IM, submucosal leiomyomas (SM), and septate uterus and fertile participants without fibroids. Initial endometrial sampling was obtained at the time of surgery, and second sampling was obtained 3 months after myomectomy/metroplasty. Expressions of each gene were evaluated using real-time reverse transcriptase polymerase chain reaction (RT-PCR). A trend toward decreased endometrial HOXA-10, HOXA-11, and ITGAV mRNA expression was detected in both SM and IM groups before myomectomy when compared to both fertile group and septate uterus. However, the differences failed to show statistical significance. After myomectomy of IM, we have detected 12.8-fold increase in endometrial HOXA-10 mRNA expression and 9.0-fold increase in endometrial HOXA-11 mRNA expression. This increase in endometrial HOXA-10 and 11 mRNA expression was significant. Accordingly, 2 patients having intramural fibroids greater than 5 cm were able to remain pregnant after myomectomy. Conversely, submucosal myomectomy did not cause any significant effect on endometrial receptivity markers. Likewise, all markers of endometrial receptivity remained unchanged after metroplasty. Myomectomy of IM have favorable effect on endometrial HOXA-10 and 11 mRNA expression.

MiRNA and target gene expression in menstrual endometria and early pregnancy decidua

ObjectiveThe role of miRNAs in modulating gene expression in decidualization remains to be determined. We performed a comparative study to identify miRNAs and their potential mRNA targets with different expression levels between endometrium and decidua. MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of the miR-146b-5p, miR-181b-5p, miR-424, miR-532, miR-199a-3p, miR-423, miR-22-3p, let-7i-5p, and miR-1 and the predicted target genes IGF2R, LEPR, SGK1, MMP2, MMP10, LIF, IL6, and STAT3 in menstrual endometria and early pregnancy decidua. ResultsmiR-146b-5p, miR-181b-5p, miR-424, miR-532, and miR-199a-3p were significantly downregulated in early pregnancy decidua, while miR-423, miR-22-3p, let-7i-5p, and miR-1 were significantly upregulated. In addition, the decidua had significantly lower levels of expression of LIF, IL6, MMP2, MMP10, and IGF2R and higher levels of expression of SGK1, LEPR, PROK1, and STAT3 than the menstrual endometria group. ConclusionOur results provide new insights into the expression of miRNAs that regulate genes involved in decidualization and the maintenance of early pregnancy.

Gamma tocotrienol targets tyrosine phosphatase SHP2 in mammospheres resulting in cell death through RAS/ERK pathway.

BackgroundThere is increasing evidence supporting the concept of cancer stem cells (CSCs), which are responsible for the initiation, growth and metastasis of tumors. CSCs are thus considered the target for future cancer therapies. To achieve this goal, identifying potential therapeutic targets for CSCs is essential.MethodsWe used a natural product of vitamin E, gamma tocotrienol (gamma-T3), to treat mammospheres and spheres from colon and cervical cancers. Western blotting and real-time RT-PCR were employed to identify the gene and protein targets of gamma-T3 in mammospheres.ResultsWe found that mammosphere growth was inhibited in a dose dependent manner, with total inhibition at high doses. Gamma-T3 also inhibited sphere growth in two other human epithelial cancers, colon and cervix. Our results suggested that both Src homology 2 domain-containing phosphatase 1 (SHP1) and 2 (SHP2) were affected by gamma-T3 which was accompanied by a decrease in K- and H-Ras gene expression and phosphorylated ERK protein levels in a dose dependent way. In contrast, expression of self-renewal genes TGF-beta and LIF, as well as ESR signal pathways were not affected by the treatment. These results suggest that gamma-T3 specifically targets SHP2 and the RAS/ERK signaling pathway.ConclusionsSHP1 and SHP2 are potential therapeutic targets for breast CSCs and gamma-T3 is a promising natural drug for future breast cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1614-1) contains supplementary material, which is available to authorized users.

Laparoscopic endometrioma resection increases peri-implantation endometrial HOXA-10 and HOXA-11 mRNA expression

To determine whether laparoscopic endometrioma resection alters peri-implantation endometrial HOXA-10, HOXA-11, LIF, ITGB3 and ITGAV mRNA expression. Case-control study. Medical school. Twenty infertile patients with uni- or bilateral endometrioma, five infertile patients having nonendometriotic benign ovarian cyst, and five fertile control subjects. Mid-luteal-phase endometrial sampling was performed at the time of surgery. Second endometrial biopsies were obtained 3 months after laparoscopic endometrioma resection during the mid-luteal phase of the cycle. Endometrial HOXA-10, HOXA-11, LIF, ITGAV, and ITGB3 mRNA expressions were evaluated with the use of reverse-transcription polymerase chain reaction. Significantly decreased endometrial ITGAV mRNA expression was noted in biopsies obtained from endometrioma and nonendometriotic cyst groups before surgery. Trends toward decreased endometrial HOXA-10, HOXA-11, LIF, and ITGB3 mRNA expressions were noted in the endometrioma and nonendometriotic cyst groups before surgery compared with the fertile subjects. However, the differences failed to show statistical significance. Compared with preoperative values, significantly increased HOXA-10 (12.1-fold change) and HOXA-11 (17.2-fold change) mRNA expressions were noted in endometrial biopsies obtained from subjects who were undergoing endometrioma surgery. Fold change in endometrial ITGAV mRNA after endometrioma surgery was found to be 30.1 and indicated a positive regulation. However, this fold increase was statistically insignificant. Expressions of these endometrial receptivity markers did not change significantly after surgical removal of nonendometriotic benign ovarian cysts. Laparoscopic endometrioma resection increases peri-implantation endometrial HOXA-10 and HOXA-11 mRNA expression, suggesting an improvement in endometrial receptivity.

Dysregulated LIF-STAT3 pathway is responsible for impaired embryo implantation in a Streptozotocin-induced diabetic mouse model.

ABSTRACTThe prevalence of diabetes is increasing worldwide with the trend of patients being young and creating a significant burden on health systems, including reproductive problems, but the effects of diabetes on embryo implantation are still poorly understood. Our study was to examine effects of diabetes on mouse embryo implantation, providing experimental basis for treating diabetes and its complications. Streptozotocin (STZ) was applied to induce type 1 diabetes from day 2 of pregnancy or pseudopregnancy in mice. Embryo transfer was used to analyze effects of uterine environment on embryo implantation. Our results revealed that the implantation rate is significantly reduced in diabetic mice compared to controls, and the change of uterine environment is the main reason leading to the decreased implantation rate. Compared to control, the levels of LIF and p-STAT3 are significantly decreased in diabetic mice on day 4 of pregnancy, and serum estrogen level is significantly higher. Estrogen stimulates LIF expression under physiological level, but the excessive estrogen inhibits LIF expression. LIF, progesterone or insulin supplement can rescue embryo implantation in diabetic mice. Our data indicated that the dysregulated LIF-STAT3 pathway caused by the high level of estrogen results in the impaired implantation in diabetic mice, which can be rescued by LIF, progesterone or insulin supplement.

Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism.

BackgroundPhotoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage.ResultsWe present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3′UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells.ConclusionsOur results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0137-1) contains supplementary material, which is available to authorized users.

The role of inherited thrombophilia and undifferentiated connective tissue dysplasia syndrome in the pathogenesis of female infertility: A clinical and morphological study

Impaired endometrial receptivity is a major cause of reproductive losses in in vitro fertilization (IVF) cycles given a normal embryo. Its causes may be associated with many diseases, including inherited thrombophilia (IT) and undifferentiated connective tissue dysplasia syndrome (uCTDS). However, endometrial receptivity remains little studied. to investigate the morphological and immunohistochemical substrates of impaired endometrial receptivity in women with uCTDS, IT, and their concurrence. Antibodies against ER, PgR, LIF, VEGF, and PAI-1 were used to morphologically and immunohistochemically examine pipelle endometrial biopsy specimens taken from 141 women in the implantation window (on days 6-7 after ovulation). In accordance with their clinical diagnoses, the patients were divided into 4 groups: 1) 13 patients with uCTDS; 2) 100 with IT; 3) 19 with uCTDS and IT; 4) 9 healthy surrogate mothers (a control group). In the examined groups, a total of 145 (90.1%) out of all the IVF protocols were unsuccessful. In the remaining 16 (9.9%) patients without exception, miscarriage started at less than 10 weeks' gestation. In the implantation window, the endometrium was immature in 101 (83.1%) women and corresponded to late proliferation or early secretion phases; 102 (84.3%) women were also found to have no mature pinopodes, pointing to the fact that the endometrial receptivity was very low. Immunohistochemical examination revealed the lower expression of the receptivity marker LIF in the endometrial surface epithelium and its higher expression in the stroma in the study groups (p < 0.05 for the uCTDS and uCTDS+IT groups) and the higher expression of PAI-1 and VEGF in the epithelium, stroma, and endothelium in the study groups than in the control group (p < 0.05), suggesting the intensity of neoangiogenetic processes and impaired fibrinolysis in these patients. uCTDS and IT are risk factors of impaired endometrial receptivity in the pathogenesis of infertility. The manifestations of impaired endometrial receptivity in this case are a decrease in mature pinopodes in the surface epithelium; focal stromal sclerosis; and redistribution of the receptivity marker LIF from the surface epithelium to the stroma, which may be used for diagnosis, prediction, and the development of targeted therapy.

Regeneration of Leydig cells in ectopically autografted adult mouse testes.

Ectopic autografting of testis tissue is a promising approach for studying testicular development, male germline preservation and restoration of male fertility. In this study, we examined the fate of various testicular cells in adult mouse testes following ectopic autografting at 1, 2, 4 and 8 weeks post grafting. Histological examination showed no evidence of re-establishment of spermatogenesis in autografts, and progressive degeneration of seminiferous tubules was detected. Expression of germ cell-specific proteins such as POU5F1, DAZL, TNP1, TNP2, PRM1 and PRM2 revealed that, although proliferating and differentiating spermatogenic germ cells such as spermatogonia, spermatocytes and spermatids could survive in autografts until 4 weeks, only terminally differentiated germ cells such as sperm persisted in autografts until 8 weeks. The presence of Sertoli and peritubular myoid cells, as indicated by expression of WT1 and ACTA2 proteins, respectively, was evident in the autografts until 8 weeks. Interestingly, seminal vesicle weight and serum testosterone level were restored in autografted mice by 8 weeks post grafting. The expression of Leydig cell-specific proteins such as CYP11A1, HSD3B2 and LHCGR showed revival of Leydig cell (LC) populations in autografts over time since grafting. Elevated expression of PDGFRA, LIF, DHH and NEFH in autografts indicated de novo regeneration of LC populations. Autografted adult testis can be used as a model for investigating Leydig cell regeneration, steroidogenesis and regulation of the intrinsic factors involved in Leydig cell development. The success of this rodent model can have therapeutic applications for adult human males undergoing sterilizing cancer therapy.

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm.

Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-588) contains supplementary material, which is available to authorized users.

Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection.

BackgroundRespiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study.MethodsA549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection.Results and discussionRSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-β). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1β, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, CXCL1, IP-10/CXCL10, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1β, IL-5, CXCL12, MIF, LIF, CXCL12 and IFN-γ. Silencing of retinoic acid–inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment.ConclusionsRSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0041-4) contains supplementary material, which is available to authorized users.