Mouse Interleukin Research Articles

Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

Induced Pluripotent Stem Cell-conditioned Medium Suppressed Melanoma Tumorigenicity Through the Enhancement of Natural-Killer Cellular Immunity.

Induced pluripotent stem cells (iPSCs) can secrete cytokines that are involved in T-cell development and affect cytotoxic activity. To assess the effect of iPSC-conditioned medium on tumorigenicity, we retrieved splenocytes from B6 mice and cocultured them with or without irradiated B16 melanoma cells, mouse interleukin-2 (mIL-2), or iPSC-conditioned medium. Splenocyte cytotoxicity assays against B16 melanoma cells [as cytotoxic T lymphocyte (CTL) activity] and P815 cells [as natural killer (NK) activity] were performed. IL-10 and interferon-γ concentrations were measured. An in vivo subcutaneous B16 melanoma growth model was performed in B6 mice and treated with iPSC-conditioned medium. The lymphocyte subpopulation depletion test was performed to determine effectors against B16 melanoma cells. We found that unstimulated splenocytes had little cytotoxic activity. Without tumor cells, mIL-2 could augment iPSC-conditioned medium-treated CTL and NK activities (P<0.01). With irradiated tumor cells, mIL-2 treatment of splenocytes could not enhance CTL or NK activity, but iPSC-conditioned medium could enhance CTL and NK activity (P<0.001). Irradiated tumor cells induced mice splenocytes to secrete more IL-10, similar to mIL-2 treatment, but not iPSC-conditioned medium treatment. mIL-2 had better efficacy than conditioned medium in inducing splenocyte interferon-γ production. The CTL and NK cell depletion test showed that the immunostimulating effect of iPSC-conditioned medium on splenocytes was through the enhancement of NK cellular activity (P<0.05). The subcutaneous melanoma growth model showed that B16-bearing mice treated with an iPSC-conditioned medium intraperitoneal injection had a decreased tumor growth rate (P<0.01). Our study suggests that iPSC-conditioned medium had a protective effect against tumor-induced immunosuppression through the enhancement of host NK cellular activity.

Mouse interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immuno/gene therapy.

Whereas cancer immunotherapy with cytokines in recent research was demonstrated effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system as well as of the Fas-mediated apoptosis by various mechanisms. We report the design and preliminary evaluation of the antitumor activity of a novel fusion protein-mIL-12/FasTI, consisting of mouse interleukin-12 and the transmembrane and intracellular domains of mouse Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase-3 activity of the clones when co-cultured with NK cells indicated that apoptosis was induced through Fas/FasL signaling pathway. The preliminary results suggest a synergized anticancer activity of the fusion protein. It may represent a promising therapeutic agent for cancer treatment.

Expression and Purification of Recombinant Mouse Interleukin-4 and -6 from Transgenic Rice Seeds

Transgenic rice seed can be utilized as a bioreactor to produce high-value recombinant proteins. Mouse interleukin 4 (mIL-4) and mIL-6 were specifically expressed as secretory proteins in rice endosperm by ligating the N-terminal glutelin B-1 (GluB-1) signal peptide and the C-terminal KDEL endoplasmic reticulum retention signal under control of the endosperm-specific GluB-1 promoter. In the transgenic rice seed, mIL-4 and mIL-6 accumulated in levels up to 0.43 mg/g grain and 0.16 mg/g grain, respectively. The reducing agents and detergents required for extraction from the transgenic rice seeds differed between the two proteins, indicating differences in their intracellular localization within the endosperm cell. Purified mIL-4 and mIL-6 exhibited high activity and very low endotoxin contamination.

Exercise-Induced Transient Increase in IL-6 Stimulates GLUT4 Expression and Enhances Insulin Sensitivity in Mouse Skeletal Muscle

A single bout of exercise induces transient increase in blood interleukin-6 (IL-6) level in human and rodents, however, the role of exercise-induced IL-6 is poorly understand. Prolonged, chronic increase in IL-6 reflects low-grade inflammation, which decrease insulin sensitivity in adipose tissue, liver and skeletal muscle. On the other hand, acute, short-period of IL-6 enhances insulin sensitivity. Because, the increase in IL-6 after exercise is transient, we hypothesized that transient increase in IL-6 after exercise enhances insulin sensitivity in skeletal muscle. C57BL6J mouse were i.v. injected normal IgG or IL-6 antibody before exercise. Twenty-four hours after a single bout of exercise (treadmill running: 20 m/min, 90 min with 10 degree incline), plantaris muscle was harvested and incubated in oxygenized KRB buffer to measure insulin-stimulated 2-deoxyglucose (2-DG) uptake. Compared with sedentary mouse, insulin-stimulated 2-DG uptake in plantaris muscle was increased 24 h after exercise in IgG-injected mouse, however, the increase induced by exercise was not observed in IL-6 antibody-injected mouse. Concomitant with this results, GLUT4 expression was increased 24 h after exercise in IgG-injected mouse, the increase was canceled in IL-6 antibody-injected mouse. Recombinant mouse IL-6 injection increased GLUT4 expression both fast-twitch plantaris muscle and slow-twitch soleus muscle in C57BL6J mouse. Furthermore, short period incubation of IL-6 (3-12 hours) increased GLUT4 expression in differentiated C2C12 myotubes, however long period (24 h) did not. These results suggests that exercise-induced transient increase in IL-6 affects skeletal muscle in autocrine/paracrine manner, which enhances GLUT4 expression leading to increase insulin sensitivity in skeletal muscle.

Interleukin-28B Plays a Therapeutic Role on Mouse U14 Cervical Cancer Cells by Down-Regulating CD4+CD25+FoxP3+Regulatory T Cells In Vivo.

To investigate the immunotherapeutic effectiveness of adenoviral vector expressing mouse interleukin (IL)-28B (Ad-mIL-28B) against cervical cancer and its mechanism. U14 cervical cancer cell-bearing mice were treated with Ad-mIL-28B. Meanwhile, whole cell vaccine was prepared by repeated freezing and thawing U14 cells. Then CD4⁺CD25⁺FoxP3⁺regulatory T (Treg) cells were evaluated by flow cytometry. Tumor volume and metastasis in BALB/c and C57BL/6j mice were detected. Ad-mIL-28B treatment significantly decreased the number of CD4⁺CD25⁺FoxP3⁺Treg cells. Subsequently, there was a significant decrease in the size of tumor tissue and the numbers of heteromorphic tumor cells. The tumor metastasis in the lung and liver of the Ad-mIL-28B group also decreased. However, there was no therapeutic effect observed for whole cell vaccine on U14 tumor-bearing mice. Interleukin-28B can inhibit the growth and metastasis of cervical cancer in U14 tumor-bearing mice by down-regulating Treg cells.

Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta.

Sinonovacula constricta has been widely used as a health food and medicine in China, Japan, and Korea. In the present study, a water-soluble polysaccharide fraction (SCP-1) was prepared from S. constricta by enzyme-assisted extraction and purification of chromatography with DEAE-52 cellulose anion-exchange column and Sephadex G-100 size exclusion column. On the basis of the analytical results of high-performance liquid chromatography, Fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, SCP-1 was found to have an average molecular weight of 15.63 kDa and a linear backbone of (1→4)-linked α-D-Glcp residue with one branch, α-D-Glcp, attached to the main chain by a (1→6) glycosidic bond at every five α-D-Glcp units. Furthermore, it was found that SCP-1 could significantly increase the viability of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β. The results suggest that SCP-1 possesses potent immunomodulating effect and may be explored as a potential biological response modifier.

Su1113 In Search of a Non-Invasive Blood Test for Diagnosis and Monitoring of EoE: A Prospective Study of a Serum Biomarker Panel

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches

Su1112 A Set of Clinical and Endoscopic Features Predicts the Presence of Eosinophilic Esophagitis With High Accuracy, Allowing Omission of Low-Yield Biopsies: Results From a Prospective Study

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches

Construction, expression, and function of 6B11ScFv-mIL-12, a fusion protein that attacks human ovarian carcinoma.

We previously produced an anti-idiotypic monoclonal antibody, 6B11, which mimics ovarian cancer antigen CA166-9 and induces cellular and humoral immunity. Here, to enhance the immunogenicity of 6B11, we constructed the 6B11ScFv-mIL-12 fusion protein (FP), by fusing single-chain fragment of 6B11 variable region (6B11ScFv) with mouse interleukin-12 (mIL-12), which was expressed in eukaryotic 293EBNA cells transfected with pSBI vectors. A binding activity assay showed 6B11ScFv-mIL-12 to have activities of both 6B11 and mIL-12-it specifically bound both ovarian monoclonal antibody COC166-9 and rabbit anti-mouse IL-12 antibody. The immune activity assay showed 6B11ScFv-mIL-12 to promote proliferation of lymphocytes stimulated by phytohemagglutinin, increase the absolute numbers and percentages of CD3(-)/CD56(+) natural killer cells and CD3(+)/CD56(+) natural killer T cells among peripheral lymphocytes, and increase interferon-γ. The FP was specifically cytotoxic to the CA166-9(+) ovarian cancer cell lines HOC1A and SKOV3 and inhibited growth of ID8 subcutaneous tumors in C57BL/6J mice. This study provides an experimental basis for clinical use of 6B11ScFv-mIL-12 in ovarian cancer therapy. To our knowledge, this is the first report of a fusion protein from an anti-idiotypic antibody and IL-12.

Establishment of cell line secreting mouse interleukin-33 and preliminary study on its anti-tumor role

Objective To establish 4T1 cell line stably secreting mouse interleukin(IL)- 33 mature protein and analyze the anti- tumor role of IL- 33 in tumor microenvironment. Methods The gene fragment encoding the mature peptide(S109 to I266)sequence of murine IL- 33 was amplified based on reverse transcriptase- polymerase chain reaction(RT- PCR)from mouse lung RNA.Then IL- 33 expression construct was generated by fusing the the fragment encoding human CD8αsignal sequence to the 5’end of IL- 33 sequence,subsequently ligated into pcDEF3 vector.The recombinant vector was transfected into 4T1 cells with LipfectamineTM 2000,and the cells were further selected with G418.Expression levels of IL- 33 in the transfectant supernatant and interferon(IFN)-γfrom stimulated CD8+ T cells by were measured by enzyme linked immunosorbent assay(ELISA). The growth and proliferation of transfectants were determined by cell counting with Trypan staining and by flow cytometry respectively. Results The stable expression of IL-33 with a concentration of 28μg/L in the supernatant of the transfected cell line was identified by ELISA.There are no differences of growth and proliferation between 4T1- vec and 4T1- IL- 33 cell lines(P> 0.05).Invitro,IL- 33 can obviously promote effector CD8+ T cell to secret IFN-γwith a concentration of 300μg/L compared with the control group 125μg/L(P<0.05).In vivo,IL- 33 secreted in the tumor microenvironment by 4T1- IL- 33 transfectant can significantly inhibit tumor growth with a tumor diameter of(6.5± 0.9)mm,compared with the control group(18.0±2.5)mm and metastasis(P<0.01). Conclusion A cell line 4T1- IL- 33 stably secreting mouse IL- 33 has been established and IL- 33 expressed in the tumor microenvironment can prominently inhibit tumor growth and metastasis. Key words: Interleukin-33; Tumor microenvironment; Effector CD8+ T; Interferon; Tumor growth and metastasis

Preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer

The purpose of this study was to examine the toxicity of combining oncolytic adenovirus-mediated cytotoxic and interleukin 12 (IL-12) gene therapy in a preclinical model to support future phase 1 trials. One hundred and twenty C57BL/6 male mice received an intraprostatic injection of saline (n = 24) or an oncolytic adenovirus (Ad5-yCD/mutTKSR39rep-mIL12) expressing two suicide genes and mouse IL-12 (n = 96). The adenovirus was administered at three dose levels (1.3 × 106, 1.3 × 107, 1.3 × 108 vp/kg) followed by 2 weeks of 5-flurocytosine (5-FC) and gancliclovir (GCV) prodrug therapy. There were no premature deaths. Daily observations of animals did not reveal any obvious clinical problems throughout the 78-day in-life phase of the study. Animals in the highest adenovirus dose group exhibited lymphopenia and transaminitis on day 3, both of which resolved by day 17. Except for mild inflammation of the prostate and seminal vesicles, histopathology of major organs was largely unremarkable. IL-12 and interferon-gamma levels in prostate and serum peaked on day 3 and were either undetectable or returned to baseline levels by day 17. No adenoviral DNA was detected in serum in any group at any time point. The results demonstrate that local administration of an oncolytic adenovirus expressing two suicide genes and IL-12 is well tolerated and support moving this investigational approach into human trials.

Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4.

BackgroundAcquisition of the M1 or M2 phenotypes by microglia has been shown to occur during the development of pathological conditions, with M1 activation being widely involved in neurotoxicity in relation with the anatomical localization and the reactivity of subtypes of microglia cells. On the contrary, little is known on the ability of microglia to undergo M2 polarization by interleukin-4 (IL4), the typical M2a polarization signal for peripheral macrophages.MethodsRecombinant mouse IL4 was injected in the third cerebral ventricle of mice to induce brain alternative polarization. The mRNA levels of Fizz1, Arg1, and Ym1 genes, known to be up-regulated by IL4 in peripheral macrophages, together with additional polarization markers, were evaluated in the striatum and frontal cortex at different time intervals after central administration of IL4; in parallel, M2a protein expression was evaluated in tissue extracts and at the cellular level.ResultsOur results show that the potency and temporal profile of IL4-mediated M2a gene induction vary depending on the gene analyzed and according to the specific brain area analyzed, with the striatum showing a reduced M2a response compared with the frontal cortex, as further substantiated by assays of polarization protein levels. Of notice, Fizz1 mRNA induction reached 100-fold level, underscoring the potency of this specific IL4 signaling pathway in the brain. In addition, immunochemistry assays demonstrated the localization of the M2 response specifically to microglia cells and, more interestingly, the existence of a subpopulation of microglia cells amenable to undergoing M2a polarization in the healthy mouse brain.ConclusionsThese results show that the responsiveness of brain macrophages to centrally administered IL4 may vary depending on the gene and brain area analyzed, and that M2a polarization can be ascribed to a subpopulation of IL4-responsive microglia cells. The biochemical pathways that enable microglia to undergo M2a activation represent key aspects for understanding the physiopathology of neuroinflammation and for developing novel therapeutic and diagnostic agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0211-6) contains supplementary material, which is available to authorized users.

Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo.

ObjectiveTo evaluate the pharmacological properties of JTE-052, a novel Janus kinase (JAK) inhibitor.MethodsThe JAK inhibitory activity of JTE-052 was evaluated using recombinant human enzymes. The inhibitory effects on cytokine signaling pathways were evaluated using primary human inflammatory cells. The in vivo efficacy and potency of JTE-052 were examined in a mouse interleukin (IL)-2-induced interferon (IFN)-γ production model and a rat collagen-induced arthritis model.ResultsJTE-052 inhibited the JAK1, JAK2, JAK3, and tyrosine kinase (Tyk)2 enzymes in an adenosine triphosphate (ATP)-competitive manner and inhibited cytokine signaling evoked by IL-2, IL-6, IL-23, granulocyte/macrophage colony-stimulating factor, and IFN-α. JTE-052 inhibited the activation of inflammatory cells, such as T cells, B cells, monocytes, and mast cells, in vitro. Oral dosing of JTE-052 resulted in potent suppression of the IL-2-induced IFN-γ production in mice with an ED50 value of 0.24 mg/kg, which was more potent than that of tofacitinib (ED50 = 1.1 mg/kg). In the collagen-induced arthritis model, JTE-052 ameliorated articular inflammation and joint destruction even in therapeutic treatments where methotrexate was ineffective.ConclusionsThe present results indicate that JTE-052 is a highly potent JAK inhibitor, and represents a candidate anti-inflammatory agent for suppressing various types of inflammation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00011-014-0782-9) contains supplementary material, which is available to authorized users.

Crystal structure of the mouse interleukin-3 β-receptor: insights into interleukin-3 binding and receptor activation.

Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of haemopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a β-subunit, the principal signalling entity. In the mouse, two β-subunits have co-evolved: a common β-subunit (βc) shared between IL-3 and the related cytokines IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF); and an IL-3-specific β-subunit (βIL-3). βIL-3 differs from βc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit, and, in the absence of structural information, the basis for these properties has remained enigmatic. In the present study, we have solved the crystal structure of the βIL-3 ectodomain at 3.45Å (1 Å=0.1nm) resolution. This structure provides the first evidence that βIL-3 adopts an arch-shaped intertwined homodimer with similar topology to the paralogous βc structure. In contrast with apo-βc, however, the ligand-binding interface of βIL-3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3-βIL-3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both βIL-3 and IL-3 underlies their unique capacity for direct interaction and specificity.

2: Generation and characterization of IL-33 cytokine traps as anti-cytokine blockers

Neutralizing the action of several cytokines with recombinant proteins corresponding to the soluble extracellular receptor chain has proven beneficial in the treatment of various autoimmune diseases in the past years. Interleukin-33 (IL-33) is a cytokine that signals through a receptor complex consisting of ST2 and IL-1RAcP, which initiates multiple pro-inflammatory signalling pathways. Blocking the IL-33/ST2 signalling axis with the soluble (extracellular) form of the ST2 receptor (sST2) has been shown to be beneficial in experimental models of asthma and arthritis. One major drawback of the use of soluble cytokine receptor proteins, however, is their relative low ligand binding affinity. Indeed, recruitment of the IL-1RAcP co-receptor upon IL-33 stimulation has been shown to significantly increase the affinity for IL-33. Here we report a new approach to interfere with IL-33 function. We have generated a fusion protein consisting of the extracellular domains of mouse ST2 and IL-1RAcP, separated by a flexible linker (referred to as IL33trap). His-tagged recombinant IL-33trap and sST2 were expressed in human HEK293T cells and purified to homogeneity from conditioned medium by metal affinity chromatography. IL-33 neutralizing activity of the purified proteins was compared in an IL-33 bioassay, in which we measure IL-33 induced NF- κ B reporter gene activation. Interestingly, the IL-33trap fusion protein is considerably more potent than sST2 in neutralizing mouse IL-33 activity. A similar approach was followed to produce an IL-33trap for human IL-33, and again the IL33trap was found to be much more potent than sST2. Together, these studies demonstrate the improved potential of IL-33trap cytokine blockers for therapeutic use. IL33trap and sST2 proteins are currently further analysed in in vitro binding affinity studies and in vivo mouse disease models.

Treatment of colon cancer by regulatable lentivirus-mediated interleukin-12 gene

Objective To evaluate the efficiency of interleukin-12 (IL-12) gene mediated by RU486 regulatable lentivirus system on tumor growth and microvessel angiogenesis of mouse colon cancer C26 cells subcutaneously implanted tumor.Methods The mouse IL-12 gene was recombined to construct the RU486 regulatable lentivirus transfer plasmid PNL-LV-RUmIL-12.The LV-RUmIL-12 lentiviruses were produced by co-transfecting PNL-LV-RUmIL-12.The expression of IL-12 in C26 colon cancer cells infected by RU486 regulated LV-RUmIL-12 lentivirus was detected by enzyme linked immunosorbent assay (ELISA) kit.Colon cancer model was established by subcutaneously inoculating C26 cells into the BALB/c mice.One hundred mice were divided into 5 groups:PBS group,blank lentivirus group (LV group),RU486 group,LV-RUmIL-12 group and LV-RUmIL-12-RU486 group.The tumor size changes were calculated.After treatment for 21 days,the mice were sacrificed by cervical dislocation.Serum levels of IL-12 were assayed by enzyme linked immunosorbent assay (ELISA).Immunohistochemical staining was employed to determine microvessel density (MVD) and CD4 + and CD8 + lymphocytes in the tumor tissues.Results After injection of LV-RUmIL-12 lentivirus,the expression of IL-12 protein in C26 cells was RU486 dose-dependent.The high-est expression level of IL-12 protein in 1 × 106 C26 cells induced by 1 × 10-5 mol/L RU486 was (148 ± 25) μg/L.Serum levels of IL-12 were (3 572.60 ±245.60),(145.43 ± 13.53),(134.53 ± 12.86),(147.32 ± 15.56) and (139.67 ± 14.84) μg/L in LV-RUmIL-12-RU486,PBS,LV,RU486 and LV-RUmIL-12 groups respectively,and IL-12 level was significantly increased in LV-RUmIL-12-RU486 group (P ＜ 0.05).The tumor growth in LV-RUmIL-12-RU486 group was markedly inhibited.The inhibition rate in LV-RUmIL-12-RU486 group was 72.59％.The MVD in LV-RUmIL-12-RU486 group (12.5 ± 4.3) was statistically decreased as copared with PBS group (45.5 ±5.7),LV group (39.5 ± 3.7),RU486 group (42.4 ±7.5) and LV-RUmIL-12 group (47.2 ±2.6,P＜0.05).AS compared with the control groups,the number of CD4 and CD8 + lymphocytes in tumor tissue of LV-RUmIL-12-RU486 group was significantly reduced (P ＜ 0.05).Conclusion The RU486 regulatable lentivirus containing IL-12 gene can inhibit the growth of transplanted mouse colon cancer in association with a significant reduction in angiogenesis. Key words: Interleukin-12; Colon cancer; Lentivirus; Angiogenesis

Long-term adaptation of the Bombyx mori BmN4 cell line to grow in serum-free culture.

Bombyx mori ovary-derived BmN4 cells have been successfully adapted to a commercial serum-free medium (SFM; SF900-II) by gradually reducing the serum-containing TC-100 medium content from 100 to 0% (v/v). The BmN4 cells adapted to the SFM (BmN-SFM) adhered strongly to the culture flask and showed altered cell morphology. The BmN-SFM was subcultured 200 times, and the population doubling time was 4.70 d. Infection studies showed that BmN-SFM cells were easily susceptible to B. mori nucleopolyhedrovirus (BmNPV), and both the multiplication of budded virus and the promoter activity of the polyhedrin gene in BmN-SFM cells were almost the same as those in BmN4 cells before adaptation. Additionally, mouse interleukin-3 expressed by a recombinant BmNPV was normally secreted and modified with N-linked glycans in BmN-SFM cells. These findings indicate that BmN-SFM is particularly useful for a BmNPV-based baculovirus expression vector system with serum-free conditions.

Platelet factor 4 inhibits IL-17/Stat3 pathway via upregulation of SOCS3 expression in melanoma.

Platelet factor 4 (PF4) was the first discovered CXC chemokine and is found in platelet granules at very high concentration. Now, it is becoming increasingly evident that PF4 actively participates in inflammation and immune response. Recent paper demonstrated that PF4 limits the development and response of the Th17 cells and assisted in regulatory T cell development in transplantation. But, the immunoregulatory role of PF4 in tumor has little known and needs to be further investigated. In our current study, wild-type mice are inoculated with melanoma cell line B16-F10 (1 × 10(6)/mouse) and treated with PF4. PF4 inhibits B16 tumor growth and decreases γδ cell infiltration. The expression of interleukin (IL)-17, IL-6, and p-signal transducer and activator of transcription-3 (Stat3) was markedly decreased with treatment of PF4 compared with control in vivo and in vitro. And, the suppressed tumor growth induced by PF4 is abolished by additional treatment of recombinant mouse IL (rmIL)-17. PF4 also induces suppressor of cytokine signaling 3 (SOCS3) upregulations, and PF4 fails to suppress expression of p-Stat3, IL-17, and IL-6 in cells transfected with SOCS3 short interfering RNA (siRNA). In conclusion, PF4 inhibits IL-17/Stat3 pathway via upregulation of SOCS3 expression and may contribute to suppressing tumor growth in murine models of melanoma.

Effects of Siglec on the expression of IL-10 in the macrophage cell line RAW264

Interleukin-10 (IL-10) expression was significantly elevated upon stimulation with lipopolysaccharide (LPS) when the sialic acid-recognizing Ig-superfamily lectin Siglec-5 or -9 was overexpressed in RAW264 cells. During the course to clarify the mechanism for this activation, we found that IL-10 promoter proximal region up to -500bp led to transactivation similar to that up to -1,500bp. Among the transcription factors that activate the mouse IL-10 promoter so far reported, the level of C/EBPβ was increased in Siglec-9-expressing cells. Transient expression of the C/EBPβ major isoform LAP led to an increase in the expression of IL-10 in Siglec-9-expressing cells, but not in mock-transfected control RAW264 cells upon stimulation with LPS, as assessed by either a luciferase assay or the production of IL-10. Without LPS, the IL-10 promoter was activated by transiently expressed LAP in Siglec-9-expressing cells, however, the magnitude of transactivation was less than that with the LPS stimulation. The knockdown of C/EBPβ down-regulated the production of IL-10. Taken together, these results suggest that one of the reasons for the stimulation of IL-10 expression in Siglec-9-expressing cells may be an increase in intracellular C/EBPβ level.