Pleiotropic Leukemia Inhibitory Factor Encapsulated in DODAB:MO Liposomes for Multiple Biomedical Applications

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.

Structural dynamics and physicochemical properties of pDNA/DODAB:MO lipoplexes: Effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases

Leukemia inhibitory factor (LIF) promotes the proliferation of neuronal progenitor cells in the cerebrum. However, it remains unclear how fetal LIF level is regulated. Here we show evidence that maternal LIF signals drive fetal LIF levels via the placenta, thereby promoting neurogenesis in the fetal brain in rats. Chronological changes showed that LIF concentration in fetal sera (FS) and fetal cerebrospinal fluid peaked at gestational day (GD) 15.5, after the peak of maternal LIF at GD14.5. LIF injection into rat dams at GD15.5 increased the level of ACTH in FS and subsequently increased LIF levels in FS and fetal cerebrospinal fluid. The elevation of fetal LIF after LIF injection into dams was inhibited by in utero injection of anti-ACTH antibody into fetuses. Cultured syncytiotrophoblasts, which express the LIF receptor and glycoprotein 130, were induced to secrete ACTH and up-regulate Pomc expression by the addition of LIF. Nucleated red blood cells from fetuses at GD15.5, but not GD13.5 or GD17.5, displayed LIF secretion in response to ACTH. Moreover, injection of LIF into dams at GD13.5 or GD17.5 did not result in elevation of ACTH or LIF in fetuses. The labeling index of 5-bromo-2'-deoxyuridine-positive cells in the ventricular zone of the cerebral neocortex increased 24 h after injection of LIF into dams at GD15.5 but not GD13.5 or GD17.5. These results suggest that in rats maternal LIF induces ACTH from the placenta, which in turn induces fetal nucleated red blood cells to secrete LIF that finally increases neurogenesis in fetuses around GD15.

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.

Leukemia Inhibitory Factor (LIF) Is Secreted by Marrow-Derived Human Mesenchymal Stem Cells (huMSC) and Inhibits Umbilical Cord Blood (UCB) Hematopoietic Stem Cell (HSC) Proliferation at Early Time Points During In Vitro Expansion.

Leukemia inhibitory factor (LIF) is a tumor promoter in several cancer types. However, the role of LIF in non-small cell lung cancer (NSCLC) remains to be explored. The present study explored the hypothesis that LIF is important for NSCLC development by measuring LIF expression and its downstream signal transducer and activator of transcription 3 (STAT3) phosphorylation in tumor samples derived from patients with NSCLC. The association between LIF expression and clinical features was analyzed in two cancer subtypes. The effects of LIF on cell proliferation, migration and invasion were also evaluated in a NSCLC-derived cell line, A549. LIF mRNA and protein expression levels were significantly higher in tumor tissues compared with those in the corresponding adjacent and normal lung tissues. Regarding NSCLC subtypes, LIF expression was significantly higher in adenocarcinoma than in squamous cell carcinoma tissues. It was also found that phosphorylated-STAT3 levels were higher in tumor tissues compared with those in the corresponding adjacent and normal lung tissues, which was in agreement with the LIF expression levels in NSCLC tissues. Clinically, overexpression of LIF was positively correlated with aggressive tumor characteristics, including lymph node metastasis and advanced tumor stage. In A549 cells, LIF treatment enhanced cell proliferation, migration and invasion. LIF also increased STAT3 phosphorylation in A549 cells, and the STAT3 inhibitor Stattic decreased A549 cell migration and invasion following LIF stimulation. The present results demonstrate that LIF is overexpressed in NSCLC, and that LIF can promote NSCLC development through activation of the STAT3 signaling pathway. The present study indicates that LIF may serve as a potential prognostic marker for NSCLC.

Leukemia inhibitory factor (LIF) is a pleiotrophic cytokine, which plays an important role in inducing cancer cachexia. We have previously reported that LIF promotes cell proliferation in some human carcinoma cells through c-fos, jun-B and cyclin-E expression. In the present study, we analyzed the regulation of LIF and its receptor (LIFR) expression in pancreatic carcinoma cells. Seven pancreatic carcinoma cells expressed constitutively LIF and its heterodimer receptor (LIFR and gp130) mRNA in RPMI-1640 medium without FBS. The amount of LIF immunoreactive protein was 132.5+/-52 pg/10(6) cells in culture supernatants without FBS. Pro-inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IL-8, or LIF, enhanced the expression of LIF mRNA in Hs-700T and Hs-766T cells. Addition of LIF significantly induced cell proliferation of Hs700T in 13 days LIF dose-dependently. However, anti-LIF IgG failed to suppress cell proliferation in Hs-700T cells. LIF acted as a paracrine growth factor in Hs-700T cells, which expressed low amount of LIF without stimuli. Cellular signal transductions by LIF was down-regulated by inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), and Ca/Calmodulin. LIF induced phosphorylation of STAT3. Moreover, exogenous LIF upregulated the expression of LIFR mRNA. Antisense LIFR oligonucleotide significantly suppressed cell growth in the presence of LIF in Hs-700T cells. These results suggest that cytokine network might alter the expression and responsiveness to LIF in tumor microenvironment.

Effects of Leukemia Inhibitory Factor on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells

Aggregation behavior of aqueous dioctadecyldimethylammonium bromide/monoolein mixtures: A multitechnique investigation on the influence of composition and temperature

We analyzed the stimulatory effect of oncostatin M (OSM), leukemia inhibitory factor (LIF), interleukin 6 (IL-6), IL-11, and the inhibitory effect of anti-IL-6 antibody (Ab), anti-IL-6 receptor monoclonal antibody (mAb), and anti-gp130 mAb on the growth of human plasmacytoma cells freshly isolated from a patient with multiple myeloma. The purified cells showed a plasmacytoid morphology and expressed CD38, CD54, and CD56 antigens but no CD3, CD5, CD10, CD19, CD20, or very late antigen 5. IL-6 receptor (IL-6R) and its signal transducer, gp130, were expressed on their cell surface at a low level. Dose-dependent proliferation of the cells in response to OSM, LIF, and IL-6, but not to IL-11, was observed using [3H]TdR incorporation in vitro. Both anti-IL-6 Ab and anti-IL-6R mAb inhibited the growth of the cells in the presence or absence of exogenous IL-6. These cells release IL-6 but not OSM or LIF into the culture supernatant during short-term culture. Therefore, an autocrine growth mechanism mediated by IL-6, but not by OSM or LIF, was confirmed. Furthermore, anti-gp130 mAb completely inhibited the proliferation of the cells induced by OSM, LIF, as well as IL-6. These data indicate that OSM, LIF, and IL-6 can act as growth factors of human plasmacytoma cells through a common signal transducer, gp130, on their cell surface, and also suggest the potential therapeutic application of anti-gp130 mAb, as well as anti-IL- 6R mAb against myeloma/plasmacytomas.

Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries