Human IL-10 Gene Research Articles

A new pipeline denoted SPICE identifies novel JUN-IKZF1 composite elements

Abstract DNA composite elements allow combinatorial transcription factor binding, which is important for fine tuning gene expression in response to physiological stimuli and in pathological conditions. Moreover, composite elements may contribute to epigenetic regulation. Accordingly, developing screening algorithms to identify novel composite elements and their compositions is crucial. Here we report a pipeline termed Spacing Preference Identification of Composite Elements (SPICE), that systematically predicts novel protein binding partners along with their DNA motifs and spacing preferences in a genome wide fashion. Besides predicting known composite elements like AP1-IRF composite elements (AICE) and STAT5 tetramers, SPICE de novo predicted a range of novel binding partners, including JUN-IKZF1 composite elements. We validated JUN-IKZF1 association by co-immunoprecipitation and showed cooperative binding of these factors to an upstream conserved noncoding region, CNS9, in the human IL10 gene based on electrophoretic mobility shift assays. Furthermore, the activity of an IL10 luciferase reporter depended on both AP1 and IKZF1 binding sites within this composite element in mouse B and T cells, confirming its transcriptional relevance. These data establish SPICE as a valuable pipeline for predicting novel transcription factor complexes that regulate gene transcription and provide insights into the biological cooperation of IKZF1 and AP1. Division of Intramural Research, NHLBI.

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) is a transcription factor essential for immunological and other biological responses. To develop analyzing system for NFAT activity in vitro and in vivo, we generated reporter mouse lines introduced with NFAT-driven enhanced green fluorescent protein (EGFP) expressing gene construct. Six tandem repeats of -286 to -265 of the human IL2 gene to which NFAT binds in association with its co-transcription factor, activator protein (AP)-1, was conjunct with thymidine kinase minimum promoter and following EGFP coding sequence. Upon introduction of the resulting reporter cassette into C57BL/6 fertilized eggs, the transgenic mice were obtained. Among 7 transgene-positive mice in 110 mice bone, 2 mice showed the designated reporter mouse character. Thus, the EGFP fluorescence of CD4+ and CD8+ T cells in these mice was enhanced by stimulation through CD3 and CD28. Each of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM) stimulation weakly but their combined stimulation strongly enhanced EGFP expression. The stimulation-induced EGFP upregulation was also observed following T cell subset differentiation in a different manner. The EGFP induction by PMA + IOM stimulation was more potent than that by CD3/CD28 stimulation in helper T (Th)1, Th2, Th9, and regulatory T cells, while both stimulation conditions displayed the equivalent EGFP induction in Th17 cells. Our NFAT reporter mouse lines are useful for analyzing stimulation-induced transcriptional activation mediated by NFAT in cooperation with AP-1 in T cells.

The NSG-hIL6 Mouse As a Novel Murine Patient Derived Xenograft Model of Plasma Cell Dyscrasias for Study of Disease Biology and Therapeutic Development

Background Plasma cell dyscrasias include pre-malignant and malignant conditions spanning the spectrum from monoclonal gammopathy of undetermined significance (MGUS) to smoldering multiple myeloma (SMM) to active multiple myeloma (aMM) and include the disorders AL amyloidosis and plasma cell leukemia (PCL). Though the last 2 decades has yielded effective therapies against these disorders, there remain no reliably curative treatments, and complications due to multiple myeloma are most often the cause of death. A major reason for the lack of curative therapies is the dearth of relevant research models in which to study these diseases and test new drug candidates. Cell lines that do not reflect the biology of mainstream myeloma are currently the mainstay of disease modeling. Plasma cells do not survive well outside the bone marrow and to this point, little success has been made using primary tumors from patients to model disease. Thus, there is a significant need for more relevant models to build the next, hopefully curative, generation of therapeutics for these diseases. Research Significance We present a novel immunodeficient murine model able to engraft plasma cell dyscrasias from primary patient tumors and the ability to study basic biology and test efficacy of cellular therapies, representing a significant advance in this field. Methods The NSG-h(IL6) mouse, developed by Dr. Shultz, builds on the background NSG mouse that lacks adaptive immunity and has limited innate immunity. It expresses a BAC with a piece of human chromosome 7, containing the human IL-6 gene and all associated local promoter and enhancer elements. With the expertise of the stem cell and xenograft core facility (SCXC), mice were preconditioned with busulfan, and 1e6 mononuclear cells (MNCs) isolated from human myeloma patient marrow aspirate were injected into the murine femur. Blood and urine were sampled every 4-5 weeks and analyzed for the presence of human antibodies by ELISA. Deceased mice were sent for histology and their spleens and marrow analyzed for the presence of clonal plasma cells by flow cytometry. Serum protein electrophoresis (SPEP) and complete blood counts (CBC) were noted as hallmarks of disease. Samples from patients with MGUS, SMM, aMM (both newly diagnosed and relapsed/refractory), PCL and AL amyloidosis were engrafted into mice as above. Mice were treated with untraduced T-cells (UTD) or BCMA-directed CARTs 10 weeks after engraftment of active myeloma and blood was assayed for level of immunoglobulins to follow response. Results Analysis of murine serum and urine by ELISA revealed the presence of rising titers of human antibodies in all engrafted mice and the presence of an M-spike on SPEP. NSG-hIL6 mice conditioned with busulfan showed greater engraftment than non-IL6 containing mice and non-conditioned mice (7 vs 2 vs 0, p<0.005). Light chain specific ELISA showed a clear predominance of one light chain, congruent with the patient sample that was engrafted. Flow cytometric analysis of engrafted murine bone marrow showed the presence of light chain restricted plasma cells. Histology showed the presence of plasma cells in the marrow, spleen and liver of engrafted animals with IHC positivity for specific light chain and CD138. CBC showed pancytopenia (p<0.005) with anemia being most significant in engrafted vs. unengrafted animals. All fresh samples and nearly all frozen samples of MGUS, SMM, aMM, PCL and AL amyloidosis were able to be engrafted at high rates (>50% of mice). Mice injected with column separated CD138+ cells alone showed no engraftment vs. mice injected with total MNCs from the marrow of the same myeloma (3 vs 0, p<0.005 ). BCMA directed CARTs significantly decreased circulating antibody compared to UTD controls (p<0.005). Conclusion Using the NSG-hIL6 mouse, busulfan conditioning and intrafemur injections, we have been able to engraft all the major plasma cell dyscrasias as denoted by the presence of monoclonal human antibody in these mice and presence of light chain restricted plasma cells congruent with the patient's paraprotein. We have preliminarily shown that the ability to engraft disease is dependent on human supporting cells. Additionally, treatment with BCMA directed CARTs is showing a response. This simple model will allow for further preclinical testing of new agents against multiple myeloma and associated plasma cell neoplasms and allow for study of basic biology of these diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Association between IL-10 rs1800872 Polymorphism in the Promoter Region of Human IL-10 Gene and Susceptibility to Rheumatoid Arthritis in the Patients in Southwest of Iran

Association between IL-10 rs1800872 Polymorphism in the Promoter Region of Human IL-10 Gene and Susceptibility to Rheumatoid Arthritis in the Patients in Southwest of Iran

In-silico analysis unravels the structural and functional consequences of non-synonymous SNPs in the human IL-10 gene

BackgroundInterleukin-10 (IL-10) is an anti-inflammatory cytokine that affects different immune cells. It is also associated with the stimulation of the T and B cells for the production of antibodies. Several genetic polymorphisms in the IL-10 gene have been reported to cause or aggravate certain diseases like inflammatory bowel disease, rheumatoid arthritis, systemic sclerosis, asthma, etc. However, the disease susceptibility and abnormal function of the mutated IL-10 variants remain obscure.ResultsIn this study, we used seven bioinformatics tools (SIFT, PROVEAN, PMut, PANTHER, PolyPhen-2, PHD-SNP, and SNPs&GO) to predict the disease susceptible non-synonymous SNPs (nsSNPs) of IL-10. Nine nsSNPs of IL-10 were predicted to be potentially deleterious: R42G, R45Q, F48L, E72G, M95T, A98D, R125S, Y155C, and I168T. Except two, all of the putative deleterious mutations are found in the highly conserved region of IL-10 protein structure, thus affecting the protein's stability. The 3-D structure of mutant proteins was modeled by project HOPE, and the protein–protein interactions were assessed with STRING. The predicted nsSNPs: R42Q, R45Q, F48L, E72G, and I168T are situated in the binding site region of the IL-10R1 receptor. Disruption of binding affinity with its receptor leads to deregulation of the JAK-STAT pathway and results in enhanced inflammation that imbalance in cellular signaling. Finally, Kaplan–Meier Plotter analysis displayed that deregulation of IL-10 expression affects gastric and ovarian cancer patients' survival rate. Thus, IL-10 could be useful as a potential prognostic marker gene for some cancers.ConclusionThis study has determined the deleterious nsSNPs of IL-10 that might contribute to the malfunction of IL-10 protein and ultimately lead to the IL-10 associated diseases.

In silico analysis of the impact of non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in the human Il-6 gene related to autoimmune diseases

In silico analysis of the impact of non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in the human Il-6 gene related to autoimmune diseases

Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells. MethodsHere the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells. ResultsThe authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut. ConclusionsCollectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.

Characterization of bioactive compounds in defatted hempseed (Cannabis sativa L.) by UHPLC-HRMS/MS and anti-inflammatory activity in primary human monocytes.

Hempseed (Cannabis sativa L.) has beneficial impact on human health mainly because of its wide variability of bioactive compounds. However, many of them are not fully characterized yet. In this work, hempseed was defatted and through a bio-guided studied, two fractions (F03 and F05) with the highest content of phenols, flavonoids and antioxidant capacity were selected. Fractions were chemically analyzed by UHPLC HRMS/MS. The anti-inflammatory capacities of these compounds were evaluated on human monocytes using flow cytometry, RT-qPCR and Elisa procedures. A high amount of phenolic compounds were identified, with the major compound being: N-trans-caffeoyltyramine (6.36 mg g-1 in F05 and 1.28 mg g-1 in F03). Both, F03 and F05 significantly reduced the inflammatory competence of LPS-treated human primary monocytes, decreasing TNF-α and IL-6 gene expression and secretion. These findings indicate that in the defatted fraction of the hempseed there are a wide number of compounds with beneficial potential to prevent and treat inflammatory disorders, as well as other processes caused by oxidative stress.

Effects of IL-10- and FasL-overexpressing dendritic cells on liver transplantation tolerance in a heterotopic liver transplantation rat model.

Acute rejection is the major determinant for the long-term survival of donor liver after liver transplantation (LT). The aim of this study was to examine the therapeutic potential of interleukin (IL)-10-FasL-overexpressing immature dendritic cells (imDCs) to induce local immunosuppression in liver grafts. imDCs derived from donors were transduced by lentiviral vectors expressing human IL-10 and/or Fas ligand (FasL) gene(s), and the expression of surface molecules and the ability to induce T-cell proliferation were measured. imDCs were intraperitoneally injected into recipient rats as a model of LT to examine the rejection grade [Banff rejection activity index (RAI)], liver functions [Alanine aminotransferase, Aspartate aminotransferase (AST) and total bilirubin (TBIL)] and post-transplant survival. IL-10 and FasL co-transduction of imDCs induced a greater reduction in CD80, CD86 and major histocompatibility complex class II (MHC II) expression, as well as T-cell proliferation, but increased levels of IL-10 and FasL in culture supernatants compared with mono-transduced or untransduced imDCs (P<0.05). The infusion of co-transduced imDCs in LT recipients reduced RAI scores, decreased plasma AST and TBIL, and prolonged survival compared with mono-transduced or untransduced imDC-treated liver allografts. These findings demonstrated that the transfusion of IL-10-FasL/imDCs enhanced immune tolerance and prolonged the survival of liver allografts after LT. The immunomodulatory activity of IL-10- and FasL-modified imDCs might be a new therapeutic approach to prevent organ rejection in clinical transplantation.

IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.

Abstract 3025: Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies

Abstract Objectives: We have developed a novel humanized IgG1 monocolonal antibody, NEO-201, which targets a neoantigen expressed on ovarian and GI malignancies. This antigen has homology to CEACAM; however, it has specificity to tumor tissues and not normal tissues. Our aims were as follows: (1) to evaluate the expression of this antigen on tumor cell lines derived from various ovarian and GI cancers; and (2) to determine the functionality of NEO-201. Methods: Expression of the antigen was identified by western blot analysis and flow cytometry. Functionality was determined by an antibody dependent cell-mediated cytotoxicity (ADCC) assay using the antigen-expressing cell lines identified by western blot analysis. Cell lines not expressing the antigen were used as negative controls. The effector cells used in the ADCC assay were natural killer (NK) cells isolated from PBMCs of normal donors and high-affinity activated NK (haNK) cells. haNK cells are NK-92 cells genetically engineered to express the high-affinity variant of human CD16 (FcgRIIIA-V158) and the human IL-2 gene. haNK cells have little or no inhibitory expression of killer cell immunoglobulin-like receptors, a unique feature that may optimize their highly cytotoxic activity against a broad range of malignancies. Results: Using western blot analysis, 2 of 5 ovarian cancer cell lines (OV90 and PE01) and 1 of 2 colorectal cancer cell lines (LS174T) stained positively for NEO-201. Flow cytometry analysis on ovarian, colorectal, and pancreatic cancer cell lines confirmed the expression of the NEO-201 neoantigen on 1 of 2 ovarian cancer cell lines (OV90), 1 of 2 colorectal cancer cell lines (LS174T), and 2 of 2 pancreatic cancer cell lines (ASPC-1 and CFPAC-1). ADCC assay was performed using OV90 and ASPC-1 tumor cell lines. Cell lines not expressing the neoantigen were used as negative controls. Killing of tumor cells expressing the neoantigen was also observed with NEO-201 alone; however, the lysis was significantly augmented when NK cells from the PBMCs of normal donors or haNK cells were included in the ADCC assay. Conclusions: These finding demonstrate that the NEO-201 neoantigen is expressed on several tumor cell lines representing ovarian and GI malignancies. NEO-201 can mediate ADCC activity in the presence of NK cells and NK cell lines. In addition, NEO-201 can induce apoptosis in tumor cell lines expressing the NEO-201 neoantigen. Furthermore, preliminary studies demonstrated that this neoantigen is expressed in numerous cancer types (not shown). In vivo studies as well as IND-related studies are planned. Citation Format: Kristen Zeligs, Philip M. Arlen, Kwong Tsang, Lidia Hernandez, Massimo Fantini, Christina M. Annunziata. Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2017-3025

NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs) are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD), which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG) mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg). After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2) cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP) or keyhole limpet hemocyanin (KLH) successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

Generation of IL10 and TGFB1 coexpressed mice displaying resistance to ovalbumin-induced asthma

Asthma is a common chronic inflammatory disease in the airways with wide prevalence, and it is thought to be caused by the combinational factors in environment and genetics. A large body of studies has suggested that cell immunity played a vital role in regulating the airway hyperreactivity (AHR) and inflammation. Therefore, we here developed a mouse model of asthma by microinjecting the pronucleus with a vector spontaneously coding human IL10 and TGFB1 gene to explore the possible interaction between these two potent molecules during asthma progression. From the total 35 newborn mice, we successfully obtained 3 founders expressing exogenous genes. In the transgenic mice, we observed profoundly enhanced expression of IL10 and TGFB1. In the condition of ovalbumin challenge, transgenic mice displayed a 1.9-fold higher MCh50 score than wild-type counterparts, indicating reminiscent AHR. Meanwhile, a three-fold decrease of cell counts in bronchoalveolar lavage fluid (BALF) was recorded as well. These results suggested that IL10 and TGFB1 cooperatively protected the respiratory system in response to antigenic stimulus. To interrogate the respective behaviors of the two genes, we quantified the expression of downstream genes in IL10 signaling or TGFB1 signaling. We observed that the examined genes in IL10 signaling were significantly repressed, especially IL5, which showed 5.4-fold decreased expression. Most genes were not altered in TGFB1 signaling, and the production of endogenous TGFB1 was significantly inhibited. These evidences collectively proved that the activation of IL0 and TGFB1 protected the host from antigen-induced asthma, possibly through IL10 signaling. This study shed some light on the modulations of IL10 and TGFB1, and related networks to asthma progression.

Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3

Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway.

STAT4-mediated transcriptional repression of the IL5 gene in human memory Th2 cells.

Type I interferon (IFN-α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/β-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/β-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model

Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma.

488. Development of a Robust Non-Viral Gene Delivery System for Gut Mucosal Cells

The gastrointestinal tract is the largest immune and endocrine organ in the body, and represents an attractive target organ for gene therapy. With a robust gene delivery system for mucosal cells, immune-modulatory proteins can be delivered locally to the gut, thus reducing the potential for systemic side effects. Furthermore, given the accessibility of the gut via the oral route, it is conceivable that a “gene pill” can be formulated to enable oral delivery of a wide range of therapeutic proteins.We report the development of a gene delivery system optimized for gut mucosal cells. The foundation of this gene delivery platform is a novel polymer, synthesized by conjugating arginine (R) and gluconic acid (GA) to low molecular weight poly-glucosamine. We demonstrated by cytotoxicity assay that this novel polymer (DDX) is biocompatible and non-toxic. Polyplex nanoparticles made from DDX containing plasmid DNA have a Z-avg diameter of ~100 nm and ζ of ~ +30 mV. In vitro transfections using DDX as a DNA carrier revealed unexpected synergistic effects, compared to mono-conjugated (R or GA) poly-glucosamine controls. In vivo intramuscular and intrarectal administration in mice also demonstrated superior gene transfection with DDX polyplex relative to R-conjugated and non-conjugated poly-glucosamine polyplexes.To demonstrate efficacy using DDX as a carrier to concentrate expression of the human IL-10 gene to the gut, we assessed the effectiveness of delivery by enema as a treatment for colitis in the dextran sulfate sodium-induced murine colitis model. Treated mice showed improved body weight recovery: (AUC: 19.3 for EG-12-treated vs 7.2 for control-treated, p=0.01). Additionally, the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-17α and TNF-α) in colon tissue of EG-12-treated mice were significantly lower than control-treated animals. Furthermore, localized delivery of IL-10 to the gut of NOD mice using the same gene delivery platform led to significant reduction in diabetes incidence, accompanied by increased regulatory T-cells and decreased activation of insulin-reactive T-cells in the mesenteric lymph nodes. Taken together, our results indicate that our novel gene transfer system can effectively deliver and concentrate immune-modulatory proteins to gut mucosal cells.We aim to formulate the polyplex for oral administration to achieve transient, but renewable, protein production in the gut. This platform may enable delivery of proteins into the circulation via the oral route. To this end, we have developed processes to convert the liquid formulation into dry powder by both spray drying or lyophilization, followed by compression into tablets or filled into capsules. Dissolution testing of these tablets and capsules in water revealed that the nanoparticles remain intact and biologically active. Preliminary studies in rats demonstrated detectable circulating IL-10 after oral administration of non-enteric coated capsules. With enteric coating, a platform capable of oral dosage of protein drugs may be made possible.

Predominant development of mature and functional human NK cells in a novel human IL-2-producing transgenic NOG mouse.

We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγ(null) (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG-IL-2 Tg). Upon transfer of human cord blood-derived hematopoietic stem cells (HSCs), CD3(-)CD56(high)CD16(+/-) cells developed unexpectedly, predominantly in the NOG-IL-2 Tg (hu-HSC NOG-IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood-derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG-IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG-IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4(+) Hodgkin's lymphoma cell line was inoculated s.c. into hu-HSC NOG-IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG-IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.

Characteristics of Viral IL-10 Gene in Epstein–Barr Virus Infection in Children in China

ABSTRACT The viral IL-10 (vIL-10) gene of Epstein–Barr virus (EBV) exhibits high homology with human IL-10 gene. In order to characterize vIL-10 gene in EBV-infected children in China, DNA sequencing of this gene was performed in 249 cases of EBV-associated infectious mononucleosis, 46 EBV-associated hemophagocytic lymphohistiocytosis and six chronic active EBV infection. Eight silent and seven nonsilent mutations were identified, of which silent mutation c9980a was prominently presented (75.7%, 228/301) in the studied cases. Two subtypes of the vIL-10 gene, including pattern B95–8 and pattern SPM (mutation in the signal peptide), were identified. Pattern B95-8 was the dominant subtype in infectious mononucleosis, EBV-hemophagocytic lymphohistiocytosis and chronic active EBV infection. These results indicated that the vIL-10 gene exists as a highly conservative form in EBV-associated infection in Chinese children.

Activation of Melanocortin Receptor 3 as a new strategy to control experimental and rheumatoid arthritis

Study ObjectiveThe anti‐inflammatory properties of melanocortin peptides have long been established, though their main molecular target(s) is still elusive. Two receptors (MC1R and MC3R) have been suggested as their main target, but the patho‐physiological impact of this important homeostatic anti‐inflammatory system is unclear: we focused here on experimental and rheumatoid (RA) arthritis.MethodsMouse Studies (1) Arthritis was induced by K/BxN serum transfer (200 μl) into wild type (WT), recessive yellow e/e (Mc1r inactive mutant) and Mc3r−/− mice and disease profiles monitored. (2) Gene expression analysis was performed in arthritic paw joints using qPCR. (3) Daily treatment with vehicle or D[Trp8]‐γ‐MSH (MC3R agonist) was given to WT and Mc3r−/− mice (20 μg) induced with K/BxN arthritis and disease profiles assessed. Human Studies qPCR was used to analyse MCR and/or pro‐inflammatory cytokine mRNA levels in RA synovium transplanted into SCID mice treated with D[Trp8]‐γ‐MSH.ResultsUpon K/BxN serum transfer, Mc3r−/− mice displayed a marked disease exacerbation whereas no different profile of disease development was displayed by the Mc1r mutant and WT mice. This heightened presence of disease in Mc3r−/− null mice was confirmed by histological analysis of paw joints. At peak of arthritic profile (D6), mRNA analysis revealed up‐regulation of Rankl, Mcp‐1, Nos2 and Il‐1β, Il‐6 in Mc3r−/− mice compared to WT. Treatment of arthritic mice with D[Trp8]‐γ‐MSH significantly attenuated disease. In another preclinical model, stimulation of human MC3R with D[Trp8]‐γ‐MSH inhibited human IL‐1β, IL‐6, TNF‐α and RANKL gene expression in RA synovium transplanted into SCID mice.ConclusionsThese findings identify MC3R‐mediated signaling as a novel anti‐inflammatory pathway for innovative therapeutic targeting in RA.