Leukemia Inhibitory Factor Levels Research Articles

Identification and genetic validation of leukemia inhibitory factor super-enhancers in acute respiratory distress syndrome and lung cancer.

Super-enhancers play prominent roles in driving robust pathological gene expression, but they are hidden in human genome at noncoding regions, making them difficult to explore. Leukemia inhibitory factor (LIF) is a multifunctional cytokine crucially involved in acute respiratory distress syndrome (ARDS) and lung cancer progression. However, the mechanisms governing LIF regulation in disease contexts remain largely unexplored. In this study, we observed elevated levels of LIF in the bronchoalveolar lavage fluid (BALF) of patients with sepsis-related ARDS compared to those with nonsepsis-related ARDS. Furthermore, both basal and LPS-induced LIF expression were under the control of super-enhancers. Through analysis of H3K27Ac ChIP-seq data, we pinpointed three potential super-enhancers (LIF-SE1, LIF-SE2, and LIF-SE3) located proximal to the LIF gene in cells. Notably, genetic deletion of any of these three super-enhancers using CRISPR-Cas9 technology led to a significant reduction in LIF expression. Moreover, in cells lacking these super-enhancers, both cell growth and invasion capabilities were substantially impaired. Our findings highlight the critical role of three specific super-enhancers in regulating LIF expression and offer new insights into the transcriptional regulation of LIF in ARDS and lung cancer.

Altered Serum Proteins Suggest Inflammation, Fibrogenesis and Angiogenesis in Adult Patients with a Fontan Circulation.

Previous omics research in patients with complex congenital heart disease and single-ventricle circulation (irrespective of the stage of palliative repair) revealed alterations in cardiac and systemic metabolism, inter alia abnormalities in energy metabolism, and inflammation, oxidative stress or endothelial dysfunction. We employed an affinity-proteomics approach focused on cell surface markers, cytokines, and chemokines in the serum of 20 adult Fontan patients with a good functioning systemic left ventricle, and we 20 matched controls to reveal any specific processes on a cellular level. Analysis of 349 proteins revealed 4 altered protein levels related to chronic inflammation, with elevated levels of syndecan-1 and glycophorin-A, as well as decreased levels of leukemia inhibitory factor and nerve growth factor-ß in Fontan patients compared to controls. All in all, this means that Fontan circulation carries specific physiological and metabolic instabilities, including chronic inflammation, oxidative stress imbalance, and consequently, possible damage to cell structure and alterations in translational pathways. A combination of proteomics-based biomarkers and the traditional biomarkers (uric acid, γGT, and cholesterol) performed best in classification (patient vs. control). A metabolism- and signaling-based approach may be helpful for a better understanding of Fontan (patho-)physiology. Syndecan-1, glycophorin-A, leukemia inhibitory factor, and nerve growth factor-ß, especially in combination with uric acid, γGT, and cholesterol, might be interesting candidate parameters to complement traditional diagnostic imaging tools and the determination of traditional biomarkers, yielding a better understanding of the development of comorbidities in Fontan patients, and they may play a future role in the identification of targets to mitigate inflammation and comorbidities in Fontan patients.

Sortilin in Biliary Epithelial Cells Promotes Ductular Reaction and Fibrosis during Cholestatic Injury

Cholestatic injuries are accompanied by a ductular reaction, initiated by proliferation and activation of biliary epithelial cells (BEC), leading to fibrosis. Sortilin (Sort1) facilitates IL-6 secretion and leukemia inhibitory factor (LIF) signaling. This study investigated the interplay between sortilin, IL-6 and LIF in cholestatic injury-induced ductular reaction, morphogenesis of new ducts and fibrosis. Cholestatic injury was induced by bile duct ligation (BDL) in WT and Sort1-/- mice, with or without augmentation of IL-6 or LIF. Mice with BEC sortilin deletion (HgfapcreSort1fl/fl) and controls were subjected to BDL and 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine diet (DDC)-induced cholestatic injury. Sort1-/- mice displayed reduced BEC proliferation and expression of BEC reactive markers. Administration of LIF or IL-6 restored BEC proliferation in Sort1-/- mice, without affecting BEC reactive or inflammatory markers. Sort1-/- mice also displayed impaired morphogenesis, which was corrected by LIF treatment. Similarly, HgfapcreSort1fl/f mice exhibited reduced BEC proliferation, but similar expression of reactive and inflammatory markers. Serum IL-6 and LIF levels were comparable, yet liver pSTAT3 was reduced, indicating that sortilin is essential for co-activation of LIFR/gp130 signaling in BEC, but not for IL-6 secretion. Notably, HgfapcreSortfl/fl mice displayed impaired morphogenesis and diminished fibrosis following BDL and DDC. In conclusion, sortilin-governed engagement of LIF signaling in BEC promotes ductular reaction and morphogenesis during cholestatic injury. Moreover, BEC sortilin is pivotal for the development of fibrosis.

IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation

BackgroundR140Q mutation in isocitrate dehydrogenase 2 (IDH2) promotes leukemogenesis. Targeting IDH2/R140Q yields encouraging therapeutic effects in the clinical setting. However, therapeutic resistance occurs in 12% of IDH2/R140Q inhibitor treated patients. The IDH2/R140Q mutant converted TF-1 cells to proliferate in a cytokine-independent manner. This study investigated the signaling pathways involved in TF-1(R140Q) cell proliferation conversion as alternative therapeutic strategies to improve outcomes in patients with acute myeloid leukemia (AML) harboring IDH2/R140Q.MethodsThe effects of IDH2/R140Q mutation on TF-1 cell survival induced by GM-CSF withdrawal were evaluated using flow cytometry assay. The expression levels of apoptosis-related proteins, total or phosphorylated STAT3/5, ERK, and AKT in wild-type TF-1(WT) or TF-1(R140Q) cells under different conditions were evaluated using western blot analysis. Cell viability was tested using MTT assay. The mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-11 in TF-1(WT) and TF-1(R140Q) cells were quantified via RT-PCR. The secretion levels of GM-CSF, OSM, and LIF were determined using ELISA.ResultsOur results showed that STAT3 and STAT5 exhibited aberrant constitutive phosphorylation in TF-1(R140Q) cells compared with TF-1(WT) cells. Inhibition of STAT3/5 phosphorylation suppressed the cytokine-independent proliferation of TF-1(R140Q) cells. Moreover, the autocrine GM-CSF, LIF and OSM levels increased, which is consistent with constitutive STAT5/3 activation in TF-1(R140Q) cells, as compared with TF-1(WT) cells.ConclusionsThe autocrine cytokines, including GM-CSF, LIF, and OSM, contribute to constitutive STAT3/5 activation in TF-1(R140Q) cells, thereby modulating IDH2/R140Q-mediated malignant proliferation in TF-1 cells. Targeting STAT3/5 phosphorylation may be a novel strategy for the treatment of AML in patients harboring the IDH2/R140Q mutation.BWcAJaJC8rGH42opvAqQCZVideo

Engineered exosome as a biological nanoplatform for drug delivery of Rosmarinic acid to improve implantation in mice with induced endometritis

Endometritis is an inflammatory and histopathologic disease in uterine tissues that interferes with the proper decidualization and implantation of the embryo. In this study, rosmarinic acid (RA) is used as an anti-inflammatory agent that encapsulates in exosomes and is used to attenuate lipopolysaccharide (LPS)-induced endometritis and improve implantation. For this purpose, exosomes were loaded with RA and then administrated into the animal groups, including RA, exosome, RA plus exosome (RA + Exo), and RA-loaded exosomes (RALExo) groups. The concentrations of RA or exosomes used in this study were 10 mg/kg, and the compounds were injected into the uterine horn 24 h following the induction of endometritis. Upon the presence of inflammation detected by the histopathological method, the most proper groups were mated with male mice. The effect of the treatment group on the implantation rate, progesterone levels, and gene expressions were assessed by Chicago Blue staining, enzyme-linked immunosorbent assay (ELISA), and Quantitative PCR (qPCR), respectively. Results showed RALExo10 and RA10 + Exo10 groups improved pathological alterations, enhanced progesterone levels, increased implantation rate, as well as heightened expression levels of Leukemia inhibitory factor (LIF) and Mucin-16 (MUC-16) genes. Besides, the expression levels of inflammatory cytokines, including Transforming growth factor-β (TGF-β), Interlukine-10 (IL-10), Interlukine-15 (IL-15), and Interlukine-18 (IL-18), were regulated. Our findings indicated that the expression of LIF, Muc-16 genes as well as IL-18, were significantly correlated with serum progesterone concentrations and the implantation rate in the treatment groups. The RALExo10 and RA10 + Exo10 groups showed ameliorated implantation rates in experimental groups.

MDM2-p53 mediate a miR-181c-3p/LIF axis to regulate low dose-rate radiation-induced DNA damage in human B lymphocytes

PurposeProlonged exposure to low dose-rate radiation (LDRR) is of growing concern to public health. Recent evidences indicates that LDRR causes deleterious health effects and is closely related to miRNAs. The aim of our study is to investigate the relationship between miRNAs and DNA damage caused by LDRR. Materials and methodsIn this study, we irradiated C57BL/6J mice with 12.5μGy/h dose of γ ray emitted from uranium ore for 8 h a day for 120 days at a total dose of 12 mGy, and identified differentially expressed miRNAs from the mice long-term exposed to LDRR through isolating serum RNAs, constructing small RNA library, Illumina sequencing. To further investigate the role of differential miRNA under LDRR，we first built DNA damage model in Immortal B cells irradiated with 12.5μGy/h dose of γ ray for 28 days at a total dose of 9.4 mGy. Then, we chose the highly conserved miR-181c-3p among 12 miRNA and its mechanism in alleviating DNA damage induced by LDRR was studied by transfection, quantitative PCR, luciferase assay, and Western blot. Results and conclusionsWe have found that 12 differentially expressed miRNAs including miR-181c-3p in serum isolated from irradiated mice. Analysis of GO and KEGG indicated that target genes of theses 12 miRNA enriched in pathways related to membrane, protein binding and cancer. Long-term exposure to LDRR induced upregulation of gamma-H2A histone family member X (γ-H2AX) expression, a classical biomarker for DNA damage in B cells. miR-181c-3p inhibited Leukemia inhibitory factor (LIF) expression via combining its 3‘UTR. LIF, MDM2, p53, and p-p53-s6 were upregulated after exposure to LDRR. In irradiated B cells, Transfection of miR-181c-3p reduced γ-H2AX expression and suppressed LIF and MDM2 protein levels, whereas p-p53-s6 expression was increased. As expected, the effect of LIF inhibition on irradiated B cells was similar to miR-181c-3p overexpression. Our results suggest that LDRR alters miRNA expression and induces DNA damage. Furthermore, miR-181c-3p can alleviate LDRR-induced DNA damage via the LIF/MDM2/p-p53-s6 pathway in human B lymphocytes. This could provide the basis for prevention and treatment of LDRR injury.

Effects of Chinese herbal medicine on pregnancy outcomes of women with PCOS undergoing in vitro fertilization and embryo transfer: A retrospective cohort study

IntroductionChinese herbal medicine is widely used for treating polycystic ovary syndrome (PCOS). The present study aimed to explore the effects of Bu-Shen-Jian-Pi Formula, a Chinese herbal medicine formula, on the pregnancy outcomes of women with PCOS undergoing in vitro fertilization and embryo transfer (IVF-ET) based on a retrospective cohort study. MethodsOne hundred and forty-six females with PCOS undergoing IVF-ET were included in the study from Women's Hospital, School of Medicine, Zhejiang University between July 2016 and June 2021, of whom 73 patients received Bu-Shen-Jian-Pi Formula treatment for 12 weeks before IVF-ET, while the other 73 did not. The clinical data were collected from the medical records of the included patients. Bone morphogenetic protein 15 (BMP15) and leukemia inhibitory factor levels in the follicular fluids were detected. ResultsAfter the treatment of Bu-Shen-Jian-Pi Formula, women with PCOS undergoing IVF-ET exhibited significant improvement in the fertilization (P<0.05), good-quality embryo (P<0.01), clinical pregnancy (P<0.01), and live birth (P<0.01) rates, and single birthweight in newborns (P<0.05). The expression levels of BMP15 in the follicular fluids of the women with PCOS were significantly increased after Bu-Shen-Jian-Pi Formula treatment (P<0.05). ConclusionThis study provided preliminary evidence of a significant association of Bu-Shen-Jian-Pi Formula treatment with improved pregnancy outcomes in women with PCOS undergoing IVF-ET, partly through increasing BMP15 levels in the follicular fluids.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity

To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. Laboratory based experimental study. Academic hospital and laboratory. Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

Leukemia Inhibitory Factor as an Indicator for Determination of the Optimal Time of Intrauterine PRP Infusion in ICSI Cases.

The compromise of uterine receptivity is one of the main reasons for the failure of assisted reproduction techniques. Leukemia inhibitory factor is one of those molecular parameters that allow us to identify an endometrium as receptive. Platelet rich plasma (PRP) contains a range of cytokines and growth factors including leukemia inhibitory factor that encourage the migration, proliferation, and differentiation of cells. Treatments for antagonist ovarian stimulation were administered to 75 women under the age of 40, and 2-D power doppler ultrasound was used to guide the oocyte harvest. Patients were allocated into three groups according to the time of intrauterine infusion of PRP (no PRP, PRP at triggering day and PRP at ova pickup day). Power Doppler is utilized to measure endometrial thickness (ET), pulsatility index (PI), and resistance index (RI) of sub endometrial arteries, as well as serum levels of leukemia inhibitory factor (LIF). The mean endometrial thickness as well as serum level of LIF in PRP trigger group were higher, resistance index and pulsatility index were lower in a significant manner than control and PRP ova pickup group at all the days of study (triggering, ova pickup and embryo transfer days). These findings revealed that intrauterine PRP infusion on ova pick up day is associated with a significant increase in serum level of LIF that in turn enhances endometrial thickness for patients scheduled for intracytoplasmic sperm injection (ICSI).

Leukemia Inhibitory Factor, Tissue Factor, Thrombomodulin, P-Selectine, D-Dimer, Thrombin Generation and Procoagulant Phospholipid Dependent Clotting Time Are Predictive for in Vitro Fertilization Outcome. the Prospective Roadmap-IVF Study

Background The link between blood hypercoagulability, endothelial cell activation and infertility or in vitro fertilization (IVF) failure is a puzzling issue. Activation of platelets, endothelial cells and blood coagulation, proinflammatory and angiogenetic potential play important role in implantation and early embryonic development. Biomarkers of cellular hypercoagulability are associated with high risk of IVF failure. Soluble Leukemia Inhibitory Factor (LIF), an interleukin 6 class cytokine expressed in the trophectoderm of the developing embryo is an important regulator in the establishment of pregnancy. Endoglin (Eng), a co-receptor for TGF-β1 and TGF-β3 is implicated in vascular complications of pregnancy and particularly in preeclampsia. Aim: The prospective longitudinal monocentric observational cohort study ROADMAP-IVF, evaluated the synergistic prognostic value of LIF and Eng with the biomarkers of cellular hypercoagulability in predicting the endometrial receptivity of fresh IVF cycles. Materials and Methods. The ROADMAP-IVF enrolled 40 women eligible for IVF with normal blood count, PT, aPTT, Fg, renal and liver function. The control group (CG) consisted of 30 healthy women with history of uncomplicated pregnancies. Exclusion criteria: Use of anticoagulant or antiplatelet agents during the last 30 days before inclusion. Known cardiovascular disease, active cancer. Active corticosteroid treatment. Primary end-point: Echographically documented pregnancy at 7 weeks from implantation. Blood was collected on day 2 (D2) from the natural cycle and 6 days after hormone treatment initiation corresponding to D8 of the cycle. Procoagulant phospholipid-dependent clotting time (PPL-ct), tissue factor (TF), thrombomodulin (TM), von Willebrand factor (vWF) and D-Dimers (DDi) were measured with assays from Diagnostica Stago (Asnieres, France). P- and E-Selectin, LIF and Eng were measured with ELISA. Thrombin generation (TG) with the TF 5 pM PPP-Reagent ® was assessed on Calibrated Automated Thrombogram (Stago, France). The cut-off values for studied biomarkers have been selected on the basis of ROC analysis. Univariate and multivariate logistic regression analysis examined the associations between the biomarkers and the study outcome. Results: 40 women were enrolled in the study. In 11 women pregnancy was confirmed with ultrasound and in 23 women no pregnancy was echographically documented. Age was not significantly different between the IVF (41 ys; range 22 - 48 ys) and the control group (40 ys; range 20 - 46 ys). At D2 and D8 the DDi and LIF were significantly higher in IVFG as compared to the CG. At D2 most of the patients had PPL-ct, DDi and MRI of TG higher than the upper normal limit (UNL). At D8 only TF was significantly increased as compared to the CG. Analytical data are presented in Table 1. Age did not correlate with the levels of the studied biomarkers. Significant correlations (p&amp;lt;0.05) were found between DDi and ETP (r=0.35); TM, TF and P-Selectin (r=0.4) at D2; TF, LIF and Eng (r=0.5) at D2 and D8; P-Selectin and PPL-ct (r=-0.4) at D2; P-Selectin and TM (r=0.4), E-Selectin (r=0.5), LIF (0.4) at D2; E-Selectin and P-Selectin (r=0.5), TF (r=0.3), and LIF (r=0.4) at D2. The ROC analysis showed that IVF failure was associate with DDi increase at D2 (AUC = 0.64); P-Selectin increase at D2(AUC = 0.57) and D8 (AUC = 0.61); TM increase at D8 (AUC = 0.61); TF increase at D8 (AUC = 0.57); LIF increase at D8 (AUC = 0.59); ETP increase at D2 (AUC = 0.67) and D8 (AUC = 0.67); Peak increase at D2 (AUC = 0.64) and D8 (AUC = 0.61). Prolongation of PPL-ct on D8 was predictive for positive pregnancy outcome(AUC 0.64). Conclusion The ROADMAP-IVF study showed that at least one out of four women eligible for IVF present biological evidence of activation of platelets, endothelial cells, blood coagulation or LIF upregulation. Hormone treatment administration does not significantly alter the proinflammatory or the hypercoagulable state. Among the studied biomarkers the levels of LIF, DDi, P-Selectine, TM and TF as well as the status of thrombin generation and PPL-ct showed a significant predictive value for the IVF outcome, particularly when measured at 6 days after hormone treatment administration. These findings will lead to the elaboration of an risk assessment model for IVF failure combining the selected biomarkers of hypercoagulability with LIF.

Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling

It is to explore, based on stromal cell derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signal axis, whether the electroacupuncture (EA) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation can promote thin endometrium regeneration and improve endometrial receptivity, so as to further study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium. Thirty matured female SD rats were randomly divided into normal control , model , BMSCs transplantation (BMSCs), BMSCs+AMD3100 (a specific antagonist of CXCR4, BMSCs+AMD3100), BMSCs+EA, and BMSCs+EA+AMD3100 groups, with 5 rats in each group. The thin endometrial model was established by intrauterine injection of 95% ethanol during the period of estrus. Rats of the model group received intravenous injection of PBS solution (tail vein) on day 1, 3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles. Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling, and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation. EA (2 Hz/15 Hz, 1 mA) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao"(SP9), "Zigong" (EX-CA1) for 15 min, once daily for 3 estrous cycles. Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension (1×106/mL) and intraperitoneal injection of AMD3100 (5 mg/kg), and those of the BMSCs+EA+AMD3100 group received administration of BMSCs, AMD3100 and EA, with both groups being once daily for 3 estrous cycles. H.E. staining was used to observe histopathological changes of endometrium tissues, and immunohistochemistry was used to detect the expressions of cytokeratin (CK19) and vimentin in endometrium (for evaluating the damage and repair of endometrium). The expression levels of homeobox A10 (HOXA10), leukemia inhibitory factor (LIF), SDF-1 and CXCR4 proteins were detected by Western blot, and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR. In comparison with the normal control group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression leve-ls of HOXA10, LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated (P<0.01), and the expression levels of SDF-1 protein and mRNA significantly up-regulated (P<0.05) in the model group. Compared with the model group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, and the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs group, and the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA, and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated (P<0.05, P<0.01). Compared to the BMSCs group, the number of endometrial glands, and the expression levels of LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated (P<0.01, P<0.05)； the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated (P<0.01). Compared to the BMSCs+EA group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated (P<0.01). Results of H.E. staining showed thin endometrium with absence of epithelial cells, and sparse glands and blood vessels, with smaller glandular cavity in the model group, which was relative milder in BMSCs and BMSCs+EA groups. EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing, thereby promoting thin endometrial regeneration, repairing endometrial injury, and improving endometrial tolerance in rats with thin endometrium.

Study the effect of recombinant leukemia inhibitory factor on maintenance of pregnancy and frequency of regulatory T cells in abortion-prone mice

Recurrent spontaneous abortion (RSA) can have a significant impact on a woman's quality of life. Understanding the mechanisms behind abortion is crucial for developing potential treatments. Among various models of abortion, the CBA/J(♀) × DBA/2J(♂) model stands out as the most extensively studied. This model reveals the influence of an altered immune system on resorption during pregnancy. The leukemia inhibitory factor (LIF) holds considerable importance as a secretory glycoprotein essential for successful implantation. Regulatory T cells (Tregs) have been found to produce high levels of LIF in both mice and humans. LIF plays a vital role in the development of Tregs by upregulating the expression of the Foxp3 transcription factor while downregulating the expression of RORγt. To investigate the impact of recombinant LIF (rLIF) on pregnancy maintenance and Treg cell frequency in abortion-prone (AP) mice, a specific recombinant protein was used in this study. The AP group consisted of CBA/J(♀) × DBA/2J(♂) mice, while the control group comprised CBA/J(♀) × BALB/c(♂) mice. Intraperitoneal injections of rLIF were administered to the AP group on the third day of pregnancy, and its effects on Treg cell frequency and pregnancy maintenance were examined during this period. Following rLIF injections on the fourteenth day of pregnancy, the expression of Foxp3 significantly increased in AP mice (p = 0.02,0.008). Additionally, AP mice injected with rLIF demonstrated a significant reduction in resorption rate (p = 0.01) and a notable increase in birth rate (p = 0.01,0.0005). These findings provide new insights into the potential benefits of LIF in treating RSA patients.

An interplay of Progesterone, Leukemia Inhibitor Factor and Interleukin-6 in the window of implantation; Impact on fertility

BackgroundThe process of implantation is crucial for the initiation of conception and hence fertility. In addition to a number of factors, it is regulated by a cross talk of gonadotrophins [Luteinizing Hormone (LH), Follicle Stimulatory Hormone (FSH)], ovarian steroids [Estrogen (Et), Progesterone (Pt)] and cytokines [Leukemia inhibitory factor (LIF) and Interleukin 6 (IL6)]. These biomarkers are chief players of implantation. ObjectiveWe aimed to explore the role of gonadotrophins (LH, FSH, LH/FSH ratio), ovarian steroids (Et, Pt) and cytokines (LIF, IL6) in the implantation process. This aim was achieved by comparing these hormones and cytokines in the fertile and infertile groups [Polycystic ovaries (PCOs), endometriosis, unexplained infertility (Uex-IF)] and finding their association in all study groups. MethodsA case control study conducted from October 2020-March 2023. A total of 135 infertile women (with PCOs, Uex-IF, and endometriosis) and 177 fertile women (matched for age and BMI) were selected. Levels of ‘Et’, ‘Pt’, ‘LIF’ and, ‘IL6′ were estimated using Enzyme Linked Immunosorbent Assay (ELISA). LH and FSH values were obtained from hospital desk records.The Independent Student'st-test was used to compare fertile and infertile groups. One-way ANOVA test was used to compare more than two groups, and Pearson’s chi-square (χ2) test was employed to compare percentages of variables. Pearson correlation analysis was performed to assess the associations and correlations. A p value < 0.05 was considered statistically significant. ResultsSignificantly higher levels of LIF and IL6 were observed in fertile women compared to infertile women. Pt levels were significantly greater in the fertile group than in the infertile group. The FSH/LH ratio was significantly higher in the fertile group. Among infertile women, PCOs (71%) and Uex-IF (91%) exhibited lower Pt levels than the fertile controls (p < 0.01), but these levels remained within the reference range (RR). Among the fertile group (81%), levels of LIF within the RR were significantly higher compared to those with Uex-IF (49%) and females with endometriosis (37%). Moreover, the highest number of participants (57%) with Uex-IF exhibited IL6 levels significantly below the RR in comparison to the fertile group and infertile groups (PCOS and endometriosis). However, lower levels of IL6 were observed in women with Uex-IF.In the control group, LIF exhibited a significant positive correlation with IL6 (r = 0.370), Pt (r = 0.496), Et (r = 0.403), and LH (r = 0.428). Among women with PCOs, LIF showed a significant positive correlation with IL6 (r = 0.443), Pt (r = 0.607), and LH (r = 0.472). In cases of Uex-IF, LIF demonstrated a significant positive correlation with IL6 (r = 0.727). Females with endometriosis displayed a significant positive correlation between LIF and IL6 (r = 0.535) as well as Pt (r = 0.605). In fertile women, a positive correlation was observed between LH and IL6 (r = 0.197, p = 0.009), LIF (r = 0.428, p = 0.000), Pt (r = 0.238, p = 0.001), and Et (r = 0.356, p = 0.000). Furthermore, a positive correlation was found between LH and LIF (r = 0.472, p = 0.000) in women with PCOs. ConclusionElevated levels of Pt were found to increase the production of LIF in fertile females. However, infertile females with PCOs and Uex-IF exhibited deficient levels of Pt, supporting its role as a biomarker for successful implantation in infertile women. These females showed decreased levels of gonadotropins as well as reduced LH/FSH ratio and diminished secretion of receptivity marker LIF, in addition to reduced Pt secretion. This suggests that reduced gonadotropin levels contribute to a lower LH/FSH ratio, resulting in decreased Pt secretion and ultimately leading to low levels of LIF, thereby causing impaired implantation in women with PCOs and Uex-IF. The exploration of low levels of LIF in patients with endometriosis requires further investigation. The significantly low levels of IL6 in the Uex-IF group elucidate the role of this cytokine in association with decreased Pt and LIF synthesis within this group.

Relation between circulating biomarkers at diagnosis of early luminal-like breast cancer and subsequent risk of distant metastases.

1077 Background: Predicting development of distant metastases after treatment of early luminal breast cancer (BC) remains a major challenge. Besides clinical-pathological features of the primary tumor, baseline circulating factors could also play a role as prognostic biomarkers. Methods: Patients with newly diagnosed early BC (grade 2/3, ER+, HER2-non- amplified) were selected from our institutional BC registry, with associated blood biobank collecting baseline plasma/serum samples at diagnosis. Circulating biomarkers potentially modulating the metastatic process (chemokines, micro-RNAs (miRNAs), Leukemia Inhibitory Factor (LIF), and osteopontin), and serum-induced functional myeloid interferon (IFN)) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling-responses, were measured at early BC diagnosis (before any treatment) in patients who subsequently developed distant metastasis within five years after diagnosis and therapy (META group) and in a 1:1 control group of patients, pairwise matched for age, tumor grade, stage, and histological subtype, who remained disease-free for at least seven years (NON-META group). All blood biomarkers were analyzed pair-wisely using the univariable Wilcoxon signed rank test (two-tailed). Multiple testing correction was done by applying a false discovery rate (FDR) of 0.05. In addition, a multivariable logistic regression analysis (MVA) was performed to find blood biomarkers signatures that can predict the development of metastases at first diagnosis of early BC. Results: 102 matched patient pairs were included. Univariable paired analysis showed significantly increased baseline expression of LIF (p=0.012) and six miRNAs (miR-143-3p: p=0.015; miR-197-3: p:&lt;0.001; miR-223-3p: p=0.006; miR-223-5p: p=0.045; miR-338-3p: p=0.045; miR-365a-3p: p=0.025), and decreased serum-induced myeloid -IFN-responses (p=0.023) and ten miRNAs (let-7b-5p: p=0.006; miR-106a-5p: p=0.045; miR-106b-5p: p=0.015; miR-107: p=0.014; miR-144-3p: p=0.015; miR-15a-5p: p=0.045; miR-15b-3p: p=0.032; miR-185-5p: p=0.045; miR-18a-5p: p=0.015; miR-30b-5p: p=0.045) in the META group compared to the NON-META group. MVA yielded a seven-biomarker signature, which includes miR-197-3p (p=0.008), miR-139-5p (p&lt;0.001), LIF (p=0.007), miR-106b-5p (p=0.003), serum-induced IFN/ISG response (p=0.006), miR-652-3p (p=0.015), and miR-133b (p=0.025) with a C-index of 0.79 (0.70-0.88) to predict distant metastases. After 5-fold internal cross-validation, C-index was 0.63 (0.52-0.74). Conclusions: Baseline circulating miRNAs, LIF, and serum-induced IFN-response levels at early luminal BC diagnosis are associated with subsequent distant metastases. Further external validation is required to confirm the prognostic value of these biomarkers.

Localized immune dysregulation is associated with lung injury in severe pediatric COVID-19

Abstract Acute respiratory distress syndrome (ARDS) is a common and devastating consequence of SARS-CoV-2 infection in adults, however severe lung injury is a rare manifestation of COVID-19 in children. Aberrant local immune responses are known to drive lung pathology in adults, but little is known about localized immune responses in children with respiratory failure due to COVID-19. We obtained blood and respiratory tract samples from children (n=35, aged 1 month to 18 years) admitted to the hospital for COVID-19, stratifying by presence of respiratory failure (n=15). We defined the cytokine and chemokine profile of severe disease using a 71 analyte Luminex panel. Children with respiratory failure were found to have increased circulating levels of leukemia inhibitory factor (LIF), a growth factor upregulated during pulmonary inflammation. The local environment revealed significant inflammation with increased levels of pro-inflammatory cytokines including IL-6, IL-8, and IL-33 among others. Application of high dimensional flow cytometry to define immune cell subsets revealed elevated proportions of monocytes in both circulating and local environments. Monocytes demonstrated decreased expression of HLA-DR and CD86, suggesting an immunoparalysis phenotype, previously associated with severe COVID-19 in adults. Further, effector memory T cells in the airway exhibited an exhausted phenotype with high levels of PD-1 expression. Together this analysis reveals a highly inflammatory and dysregulated immune response underlies SARS-CoV-2 induced pediatric lung injury. These findings suggest a shared pathway with adult COVID-19 ARDS and provide new insights into the immune mechanisms associated with severe COVID-19 in children. Supported by grants from NIH (K23AI141686)

Association between Cardiovascular Response and Inflammatory Cytokines in Non-Small Cell Lung Cancer Patients

Cardiovascular disease is an essential comorbidity in patients with non-small cell lung cancer (NSCLC) and represents an independent risk factor for increased mortality. Therefore, careful monitoring of cardiovascular disease is crucial in the healthcare of NSCLC patients. Inflammatory factors have previously been associated with myocardial damage in NSCLC patients, but it remains unclear whether serum inflammatory factors can be utilized to assess the cardiovascular health status in NSCLC patients. A total of 118 NSCLC patients were enrolled in this cross-sectional study, and their baseline data were collected through a hospital electronic medical record system. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of leukemia inhibitory factor (LIF), interleukin (IL)-18, IL-1β, transforming growth factor-β1 (TGF-β1), and connective tissue growth factor (CTGF). Statistical analysis was performed using the SPSS software. Multivariate and ordinal logistic regression models were constructed. The data revealed an increased serum level of LIF in the group using tyrosine kinase inhibitor (TKI)-targeted drugs compared to non-users (p < 0.001). Furthermore, serum TGF-β1 (area under the curve, AUC: 0.616) and cardiac troponin T (cTnT) (AUC: 0.720) levels were clinically evaluated and found to be correlated with pre-clinical cardiovascular injury in NSCLC patients. Notably, the serum levels of cTnT and TGF-β1 were found to indicate the extent of pre-clinical cardiovascular injury in NSCLC patients. In conclusion, the results suggest that serum LIF, as well as TGFβ1 together with cTnT, are potential serum biomarkers for the assessment of cardiovascular status in NSCLC patients. These findings offer novel insights into the assessment of cardiovascular health and underscore the importance of monitoring cardiovascular health in the management of NSCLC patients.

Abstract 4966: Targeting LIF/LIFR autocrine loops with EC359 in ovarian cancer: A novel LIFR targeted therapy

Abstract Background: Of all gynecologic cancers, ovarian cancer (OCa) has the highest mortality rates. Nearly 90% of patients who receive standard surgical and cytotoxic treatment experience disease recurrence. Leukemia inhibitory factor (LIF) and its receptor LIFR are implicated in the progression of several cancers. A knowledge gap exists on whether LIF/LIFR plays a role in the evolution of OCa. We recently developed EC359, a first-in-class LIFR inhibitor. Here, we examined whether autocrine loops of LIF/LIFR contribute to OCa progression and tested the utility of EC359 as a potential targeted therapy. Methods: Eighteen different OCa model cells, both established and primary, were used to profile the expression of LIF and LIFR. Cell viability, colony formation, apoptosis, and reporter assays were used to assess EC359 impact on OCa cells. Mechanistic studies were carried out using RNA-seq and RT-qPCR analysis. Using cell-based xenografts, syngeneic xenografts, patient derived organoids (PDO), and patient derived xenograft (PDX) models, the effectiveness of LIFR inhibitor EC359 as a targeted therapy was examined. Results: Kaplan-Meier survival analysis (KMplot) revealed increased expression of LIF and LIFR was linked to poor progression-free survival in OCa patients. The levels of LIF and LIFR were considerably greater in OCa chemotherapy non-responders than responders. We validated the existence of LIF/LIFR autocrine signaling using 18 distinct OCa cells. Treatment with the LIFR inhibitor EC359 dramatically decreased OCa cell viability, cell survival and increased apoptosis, with an IC50 of 5 to 50 nM. The activation of STAT3, mTOR, AKT, and p42/44 MAPKs as well as other downstream LIFR signaling was markedly decreased by EC359 treatment. Treatment with EC359 also decreased the stemness of OCa cells, slowed PDO development, and sensitized chemotherapy-resistant OCa cells to chemotherapy. One of the significant pathways elevated by EC359, according to RNA-seq data, is the regulation of apoptosis. In six different cell-based xenografts and PDX tumors, we demonstrated that the EC359 at 5mg/kg dose significantly reduced the OCa xenograft growth. In comparison to the vehicle control, the tumor volume was significantly reduced by EC359 treatment of murine ID8 xenografts in C57BL6 mice. Our findings indicated that EC359 had both intrinsic and extrinsic effects on tumors. Tumor-associated macrophages (TAMs) with a significant M1 polarity (CD11b+Gr1-CD68high/phosphoSTAT1+/cMAF-) and robust tumor infiltration by (CD45+) leukocytes were enhanced with EC359 therapy of ID8 xenograft tumors. Importantly, normal T, B, and other immune cells in the blood demonstrated that EC359 had no effect on immune cell homeostasis. Conclusions: Together, our findings support the existence of LIF/LIFR autocrine loops, and EC359 is a viable treatment option for OCa. Citation Format: Behnam Ebrahimi, Suryavathi Viswanadhapalli, Uday P. Pratap, Rahul Gopalam, Xue Yang, Bindhu Santhamma, Swapna Konda, Xiaonan Li, Hui Yan, Gangadhara R. Sareddy, Zhenming Xu, Edward R. Kost, Rajeshwar R. Tekmal, Hareesh B. Nair, Ratna K. Vadlamudi. Targeting LIF/LIFR autocrine loops with EC359 in ovarian cancer: A novel LIFR targeted therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4966.

Effect of acupuncture on the opening time of implantation window and endometrial receptivity in controlled ovarian hyperstimulation rats during peri-implantation period.

To investigate the effect of acupuncture for improving the pregnancy rate of COH rats from the viewpoint of regulating the opening time of the implantation window and endometrial receptivity. Experimental rats were randomly divided into normal group (N), model group (M) and acupuncture group(A), and samples were collected on Day 4, 5 and 6 after mating. COH rats were treated with acupuncture at SP6, LR3, and ST36 once a day for 7 times. The pinopodes were observed under a scanning electron microscope. Serum estrogen and progesterone levels were measured via ELISA. The protein and mRNA levels of estrogen receptor (ER), progesterone receptor (PR), leukemia inhibitory factor (LIF), integrin β3, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) in the endometrium were evaluated via West-blot, immunohistochemistry, and PCR. Compared with group N, the pregnancy rate of group M was significantly decreased (P<0.05), and the abnormal serum hormone levels and implantation window advancement were observed. Compared with group M, the pregnancy rate of group A was significantly increased (P<0.05), the supraphysiological serum progesterone levels were restored to normalcy (P<0.05), and the advanced implantation window was restored to a certain extent. Further, the abnormal ER, PR, LIF, integrin β3, VEGF, and FGF-2 expression levels of the endometrium got recovered to varying degrees. Acupuncture may restore the estrogen and progesterone balance in COH rats and the forward shift of the implantation window to a certain extent, improving the endometrial receptivity and finally improving the pregnancy rate of COH rats.

Possible Role of Leukemia Inhibitory Factor and Inflammatory Cytokines in The Recurrent Spontaneous Abortion: A Case-Control Study.

Despite of long-lasting efforts, in more than 50% of cases, the etiology of recurrent spontaneous abortion (RSA) remains unknown. Leukemia inhibitory factor (LIF) has an essential role in the reproductive process, such as modulating inflammatory responses. This study aimed to evaluate the relationship between the LIF gene expression as well as serum levels of inflammatory cytokines and occurrence of RSA in infertile women with a history of RSA. In this case-control study, the relative gene expression levels of LIF, concentrations of tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-17 were measured in peripheral blood and serum of women with a history of RSA (N=40) compared with non-pregnant and fertile women as the control group (N=40) using quantitative real-time polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. The mean age of patients and controls was 30.1 ± 4.28 and 30.03 ± 4.23, respectively. Patients had a history of at least 2 and at most 6 abortions. The mRNA levels of LIF were significantly lower in the women with RSA in comparison with the healthy participant (P=0.003). Regarding cytokine levels, no significant difference was seen between the two groups (P≥0.05). There was no correlation - between the LIF mRNA levels and TNF-α and IL-17 serum concentrations. The U-Mann-Whitney test and the Pearson correlation coefficient were applied to comparison variables between groups as well as a correlation between LIF mRNA and cytokine levels in serum. Despite a significant reduction in the LIF gene mRNA level in patients with RSA, it was not associated with increases in inflammatory cytokines. Dysfunction in the production of LIF protein may be involved in the onset of RSA disorder.

Oil-Based Hysterosalpingography ?mproves Leukaemia ?nhibitory Factor Expression ?n Endometrial Flushing Samples

Objective: The biological mechanism of the fertility-enhancing effect of hysterosalpingography (HSG) is not fully known. Leukemia inhibitory factor (LIF) is an important cytokine involved in implantation. This study was planned to investigate the effect of HSG on LIF mRNA expression in endometrial flushing samples. Methods: Forty infertile patients scheduled for HSG were included in the study. Before HSG, endometrial flushing was performed in the mid-luteal phase, followed by contrast-medium infusion into the cavity. Second flushing was done in the mid-luteal phase of the next cycle. Ten patients who underwent diagnostic hysteroscopy were taken as the second control group. LIF mRNA levels were measured by RT-PCR in endometrial flushing samples collected from each group. Results: The pre-HSG LIF mRNA levels of the infertile group were significantly lower than those of the fertile group. In infertile patients, LIF mRNA expression increased 3.2 times after HSG compared to baseline levels. LIF values reached fertile levels after HSG. While the LIF levels measured in the hysteroscopy group were significantly lower than in fertile patients, they were similar to the pre-HSG values of infertile patients. LIF levels of infertile patients after HSG were found to be significantly higher than those of the hysteroscopy group. Conclusions: In infertile patients, HSG contributes to receptivity by increasing LIF mRNA synthesis.