Science.gov

Sample records for gun-mediated il-12 gene

  1. A Novel Gene Gun-Mediated IL-12 Gene Therapy for Breast Cancer

    DTIC Science & Technology

    1999-01-01

    gene therapy . The results of the first year study, described in our previous Annual Report, show that gene gun-mediated IL-12 gene therapy is effective against breast tumors in mouse models. During the second year of this study we demonstrated that 4T1 tumor is weakly immunogenic, and it can induce a low level immune response. However, the anti-metastatic effect of IL-12 gene therapy against 4T1 tumor is not mediated by T cells, but rather involves NK cells. From several different immunomidulatory genes tested in combination with

  2. A Novel Gene Gun-Mediated IL-12 Gene Therapy for Breast Cancer

    DTIC Science & Technology

    2000-10-01

    The results of this study show that particle-mediated IL-12 gene therapy was effective against mammary tumors in mouse models. IL-12 gene therapy of...combination with IL-12 gene therapy , IL-18 and ICE genes were found to be more effective in treatment of established TS/A mammary tumor than IL-12 alone. These...results suggest that particle-mediated IL-12 gene therapy , alone or in combination with other immunological approaches, may be effective for

  3. A Novel Gene Gun-Mediated IL-12 Gene Therapy for Breast Cancer

    DTIC Science & Technology

    1999-10-01

    The overall goal of our research is to develop an immunological approach for breast cancer gene therapy . The results of the first year study...described in our previous Annual Report, show that gene gun-mediated Th-12 gene therapy is effective against breast tumors in mouse models. During the second...effect of IL-l2 gene therapy against 4T1 tumor is not mediated by T cells, but rather involves NK cells. From several different immunomodulatory genes

  4. A Novel Gene Gun-Mediated IL-12 Gene Therapy for Breast Cancer

    DTIC Science & Technology

    1997-10-01

    immunogenic 4T1 tumor, primary tumor growth was not affected by IL-12 gene therapy , although lung metastasis was significantly reduced. The anti...metastatically effect in the 4T1 model appears to be T cell independent, and we are investigating its mechanism. These results suggest that a similar gene therapy protocol may be useful in human breast cancer treatment.

  5. IL-12 based gene therapy in veterinary medicine.

    PubMed

    Pavlin, Darja; Cemazar, Maja; Sersa, Gregor; Tozon, Natasa

    2012-11-21

    The use of large animals as an experimental model for novel treatment techniques has many advantages over the use of laboratory animals, so veterinary medicine is becoming an increasingly important translational bridge between preclinical studies and human medicine. The results of preclinical studies show that gene therapy with therapeutic gene encoding interleukin-12 (IL-12) displays pronounced antitumor effects in various tumor models. A number of different studies employing this therapeutic plasmid, delivered by either viral or non-viral methods, have also been undertaken in veterinary oncology. In cats, adenoviral delivery into soft tissue sarcomas has been employed. In horses, naked plasmid DNA has been delivered by direct intratumoral injection into nodules of metastatic melanoma. In dogs, various types of tumors have been treated with either local or systemic IL-12 electrogene therapy. The results of these studies show that IL-12 based gene therapy elicits a good antitumor effect on spontaneously occurring tumors in large animals, while being safe and well tolerated by the animals. Hopefully, such results will lead to further investigation of this therapy in veterinary medicine and successful translation into human clinical trials.

  6. Gene therapy with IL-12 induced enhanced anti-tumor activity in fibrosarcoma mouse model.

    PubMed

    Razi Soofiyani, Saiedeh; Kazemi, Tohid; Lotfipour, Farzaneh; Mohammad Hosseini, Akbar; Shanehbandi, Dariush; Hallaj-Nezhadi, Somayeh; Baradaran, Behzad

    2016-12-01

    Context Immunotherapy is among the most promising modalities for treatment of cancer. Recently, interleukin 12 (IL-12) has been used as an immunotherapeutic agent in cancer gene therapy. IL-12 can activate dendritic cells (DCs) and boost anti-tumor immune responses. Objective In the current study, we have investigated if IL-12 gene therapy can lead to the regression of tumor mass in a mouse model of fibrosarcoma. Material and methods To investigate the therapeutic efficacy of IL-12, WEHI-164 tumor cells were transfected with murine-IL12 plasmids using Lipofectamine. Enzyme linked immunosorbent assay (ELISA) was used to confirm IL-12 expression in transfected cells. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/C mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of IL-12 and IFN-γ was studied with real-time PCR and western blotting. The expression of Ki-67(a tumor proliferation marker) in tumor mass was studied by immunohistochemistry staining. Results and discussion The group treated with IL-12 showed a significant decrease in tumor mass volume (P: 0.000). The results of real-time PCR and western blotting showed that IL-12 and IFN-γ expression increased in the group treated with IL-12 (relative expression of IL-12: 1.9 and relative expression of IFN-γ: 1.766). Immunohistochemistry staining showed that Ki-67 expression was reduced in the group treated with IL-12. Conclusion IL-12 gene therapy successfully led to regress of tumor mass in the fibrosarcoma mouse model. This may serve as a candidate therapeutic approach for treatment of cancer.

  7. Combined Tbet and IL12 gene therapy elicits and recruits superior antitumor immunity in vivo.

    PubMed

    Qu, Yanyan; Chen, Lu; Lowe, Devin B; Storkus, Walter J; Taylor, Jennifer L

    2012-03-01

    We have recently shown that intratumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 complementary DNA (cDNA) would yield improved antitumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established subcutaneous (s.c.) tumors injected with DC concomitantly expressing ectopic Tbet and IL12 (i.e., DC.Tbet/IL12) displayed superior (i) rates of tumor rejection and extended overall survival, (ii) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and (iii) infiltration of CD8(+) T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translation use of combined Tbet and IL-12p70 gene therapy in the cancer setting.

  8. Gene therapy using IL 12 family members in infection, auto immunity, and cancer.

    PubMed

    Waldner, Maximilian J; Neurath, Markus F

    2009-08-01

    Interleukin-12 (IL-12) is known for several years to have an essential role in inflammatory responses and innate resistance to infection and cancer. This has been largely attributed to its ability to initiate the differentiation of T-helper-1 (Th1) cells producing interferon-gamma. Recently, two new cytokines, IL-23 and IL-27, with homology to IL-12 were discovered and assigned to the IL-12 family of cytokines. Growing evidence supports a role for IL-23 as key mediator of autoimmune disease regulating the new Th17 subset of CD4+ T cells. IL-27 can have pro- and anti-inflammatory effects, which increase Th1 differentiation, suppress Th2 proliferation, or stimulate cytotoxic T cell activity. Several strategies have been pursued to apply the immunological effects of IL-12 family members to the treatment of human disease. Whereas the inhibition of IL-12 and IL-23 signal transduction has shown promising results for the treatment of autoimmune disease, the administration of IL-12 during infection and cancer can increase the host immune reaction. The increasing knowledge about the new IL-12 family members, IL-23 and IL-27, has revealed new therapeutic options for the use of these cytokines. In this review, we discuss therapeutic strategies using IL-12 family members in infection, autoimmunity, and cancer with special focus on gene administration.

  9. Analysis of IL12B Gene Variants in Inflammatory Bowel Disease

    PubMed Central

    Wagner, Johanna; Olszak, Torsten; Fries, Christoph; Tillack, Cornelia; Friedrich, Matthias; Beigel, Florian; Stallhofer, Johannes; Steib, Christian; Wetzke, Martin; Göke, Burkhard; Ochsenkühn, Thomas; Diegelmann, Julia; Czamara, Darina; Brand, Stephan

    2012-01-01

    Background IL12B encodes the p40 subunit of IL-12, which is also part of IL-23. Recent genome-wide association studies identified IL12B and IL23R as susceptibility genes for inflammatory bowel disease (IBD). However, the phenotypic effects and potential gene-gene interactions of IL12B variants are largely unknown. Methodology/Principal Findings We analyzed IL12B gene variants regarding association with Crohn's disease (CD) and ulcerative colitis (UC). Genomic DNA from 2196 individuals including 913 CD patients, 318 UC patients and 965 healthy, unrelated controls was analyzed for four SNPs in the IL12B gene region (rs3212227, rs17860508, rs10045431, rs6887695). Our analysis revealed an association of the IL12B SNP rs6887695 with susceptibility to IBD (p = 0.035; OR 1.15 [95% CI 1.01–1.31] including a trend for rs6887695 for association with CD (OR 1.41; [0.99–1.31], p = 0.066) and UC (OR 1.18 [0.97–1.43], p = 0.092). CD patients, who were homozygous C/C carriers of this SNP, had significantly more often non-stricturing, non-penetrating disease than carriers of the G allele (p = 6.8×10−5; OR = 2.84, 95% CI 1.66–4.84), while C/C homozygous UC patients had less often extensive colitis than G allele carriers (p = 0.029; OR = 0.36, 95% CI 0.14–0.92). In silico analysis predicted stronger binding of the minor C allele of rs6887695 to the transcription factor RORα which is involved in Th17 differentiation. Differences regarding the binding to the major and minor allele sequence of rs6887695 were also predicted for the transcription factors HSF1, HSF2, MZF1 and Oct-1. Epistasis analysis revealed weak epistasis of the IL12B SNP rs6887695 with several SNPs (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694) in the STAT4 gene which encodes the major IL-12 downstream transcription factor STAT4 (p<0.05) but there was no epistasis between IL23R and IL12B variants. Conclusions/Significance The IL12B SNP rs6887695 modulates

  10. Anti-tumor mechanism in IL-12 Gene therapy using liposomal bubbles and ultrasound

    NASA Astrophysics Data System (ADS)

    Suzuki, Ryo; Oda, Yusuke; Koshima, Risa; Hirata, Keiichi; Nomura, Tetsuya; Negishi, Yoichi; Utoguchi, Naoki; Nakagawa, Shinsaku; Maruyama, Kazuo

    2011-09-01

    Sonoporation combined with nano/microbubbles is an attractive technique for developing non-invasive and non-viral gene delivery systems. Previously, we developed novel ultrasound sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. IL-12 corded plasmid DNA delivery into tumor tissue by sonoporation combined with Bubble liposomes was found to suppress tumor growth. In this study, we examined the mechanism of the anti-tumor effect in this IL-12 gene delivery. This therapeutic effect was T-cell dependent, requiring mainly CD8+ T lymphocytes in the effector phase, as confirmed by a mouse in vivo depletion assay. In addition, migration of CD8+ T cells was observed in the mice. These results suggest that CD8+ T lymphocytes play an important role in the anti-tumor effects of this IL-12 gene therapy.

  11. Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

    PubMed

    Kim, Jung Seok; Kang, Seong Jae; Jeong, Hwa Yeon; Kim, Min Woo; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

    2016-09-01

    Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

  12. IL-10 and IL-12B gene polymorphisms in a multiethnic Malaysian population.

    PubMed

    Sam, S S; Teoh, B T; AbuBakar, S

    2015-04-13

    Inheritance of polymorphisms in the interleukin (IL)-10 promoter and IL-12B genes, which influence cytokine production and activities, may define the balance in T helper response in infection and autoimmune diseases. In the present study, we investigated the distribution of the IL-10 promoter and IL-12B gene polymorphisms in a multiethnic Malaysian population. Overall, our findings suggest that the IL-12B and IL-10 -592 genotypes were distributed homogenously across all major ethnic groups, including Malays, Chinese, and Indians, except for polymorphisms at IL-10 -1082. At this gene locus, the ethnic Chinese showed a significantly lower allele frequency of -1082G (2.1%) compared to the Malay (12.2%) and Indian (15.3%) populations. Results for the IL-12B and IL-10 gene polymorphisms were consistent with those reported for the Asian population, but markedly different from those of the African and Caucasian populations. Our findings suggest that there are specific genetic variations between different ethnic groups, which should be examined in all gene population-based association studies.

  13. Haplotype analysis finds linkage disequilibrium in the IL-12 gene in patients with HCV.

    PubMed

    Houldsworth, Annwyne; Metzner, Magdalena; Hodgkinson, Andrea; Shaw, Steve; Kaminski, Edward; Demaine, Andy G; Cramp, Matthew E

    2015-07-01

    HCV is a major cause of liver disease worldwide. IL-12 plays an essential role in the balance of T helper 1 (Th1) differentiation versus a T helper 2 (Th2) driven response from its naïve precursor. Linkage disequilibrium measures the degree to which alleles at two loci are associated and the non-random associations between alleles at two loci. Haplotypes of the three IL-12B loci studied were determined in the patient cases and the normal healthy control subjects. The frequency of the 12 possible IL-12B haplotypes on the 3 loci was determined in subjects heterozygous at only one of the loci within the studied haplotype. Haplotype frequencies were compared between the patient groups and controls (n = 49) to determine if any preferential combination of markers occurred using chi-squared and applying the Bonferroni correction. 45 HCV RNA negative patients; 88 HCV RNA positive patients; and 15 uninfected cases at high risk of HCV infection (EU) were studied. The haplotype "C" SNP of the 3'UTR with the "E" 4 bp deletion of the intron 4 region was in linkage disequilibrium (χ(2)  = 45.15, P < 0.001, 95% CL). The haplotype analysis of the insertion allele of the promoter with the deletion allele of the intron 4("E") IL-12B polymorphism showed linkage disequilibrium (χ(2)  = 5.64, P = 0.02). Linkage disequilibrium of polymorphisms is reported in the IL-12 gene in patients with HCV infection and contributes to the understanding of patient genotype and expected production of IL-12, responding to infection. © 2015 Wiley Periodicals, Inc.

  14. Impact of obesity on IL-12 family gene expression in insulin responsive tissues.

    PubMed

    Nam, Heesun; Ferguson, Bradley S; Stephens, Jacqueline M; Morrison, Ron F

    2013-01-01

    Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages.

    PubMed

    Chandran, Aneesh; Antony, Cecil; Jose, Leny; Mundayoor, Sathish; Natarajan, Krishnamurthy; Kumar, R Ajay

    2015-01-01

    Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live, but not killed, virulent MTB. Additionally, we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live, virulent MTB, and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases, and by knockdown of crucial transcriptional regulators, we demonstrate that protein kinase-A (PKA), CREB, and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis, we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live, virulent MTB to subvert the immune system to survive and replicate in the host.

  16. Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

    PubMed Central

    Cutrera, Jeffry; Dibra, Denada; Xia, Xueqing; Hasan, Azeem; Reed, Scott; Li, Shulin

    2011-01-01

    Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments. PMID:21386825

  17. Definition of polymorphisms and haplotypes in the interleukin-12B gene: association with IL-12 production but not with Crohn's disease.

    PubMed

    Zwiers, A; Seegers, D; Heijmans, R; Koch, A; Hampe, J; Nikolaus, S; Peña, A S; Schreiber, S; Bouma, G

    2004-12-01

    Interleukin-12 (IL-12) is a key cytokine for the induction of Th1 immune responses. Recently, functional polymorphisms in IL-12p40 (IL12B) were found to be associated with susceptibility to several autoimmune diseases. Similarly, variation in IL12B might be involved in susceptibility to Crohn's disease (CD), a chronic inflammatory bowel disorder associated with high IL-12 expression. We searched for additional polymorphism in IL12B and genotyped a large cohort of CD patients. Differential in vitro secretors of IL-12 were tested for polymorphism. Polymorphisms were analyzed using the intrafamilial transmission disequilibrium test (TDT) and by case-control analysis. A novel polymorphism was strongly associated with differential expression of IL-12. However, no association with susceptibility to CD was seen for this and other polymorphisms. The high level of conservation is consistent with the key regulatory role of IL-12. The lack of association with IL12B makes it unlikely that this gene is directly involved in the susceptibility to CD.

  18. The relationship between the antitumor effect of the IL-12 gene therapy and the expression of Th1 cytokines in an HPV16-positive murine tumor model.

    PubMed

    García Paz, Flor; Madrid Marina, Vicente; Morales Ortega, Ausencio; Santander González, Abimelec; Peralta Zaragoza, Oscar; Burguete García, Ana; Torres Poveda, Kirvis; Moreno, José; Alcocer González, Juan; Hernandez Marquez, Eva; Bermúdez Morales, Victor

    2014-01-01

    The goal of the present study was to investigate the effect of IL-12 expressed in plasmid on the Th1 cytokine profile in an experimental HPV16-positive murine tumor model and the association with the IL-12's antitumor effect. Mice were injected with BMK-16/myc cells to establish HPV16-positive tumor and then pNGVL3-mIL-12 plasmid; pcDNA3 plasmid or PBS was injected directly into tumor site. The antitumor effect of the treatment was evaluated and the cytokines expression profile in each tumor tissue was analyzed. Treatment with pNGVL3-mIL-12 plasmid had a significant antitumor effect, and a Th2-Th3-type cytokines prolife was detected in the murine tumor model with expression of the cytokines IL-10, IL-4, and TGF-β1. However, after the tumor was treated with three intratumoral injections of plasmid containing IL-12 cDNA, it showed a cytokine profile associated with Th1 with expression of IL-2, IL-12, and IFN-γ cytokines and reduced expression of IL-10, IL-4, and TGF-β1. The treatment with the IL-12 gene in the experimental HPV16-positive tumor model promoted the activation of the cellular immune response via expression of a Th1-type cytokine profile and was associated with the inhibition of tumor growth. Thus, IL-12 treatment represents a novel approach for gene therapy against cervical cancer.

  19. Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum).

    PubMed

    Gondo, Takahiro; Tsuruta, Shin-ichi; Akashi, Ryo; Kawamura, Osamu; Hoffmann, Franz

    2005-12-01

    We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.

  20. Antitumoral activity of transferrin-lipoplexes carrying the IL-12 gene in the treatment of colon cancer.

    PubMed

    Tros De Ilarduya, Conchita; Buñuales, Maria; Qian, Cheng; Düzgüneş, Nejat

    2006-09-01

    The present study aimed to establish an efficient targeted nonviral strategy for IL-12 gene transfer in colon carcinoma in vivo employing transferrin (Tf)-lipoplexes. Complexes for in vitro experiments were prepared at a 5/1(+/ - ) (lipid/DNA) charge ratio, with the ligand Tf (32 (microg/(microg DNA). Complexes for in vivo experiments contained 144 mM of total lipid (DOTAP/Chol), 60 (microg of pCMVLuc or pCMVIL-12 and 32 (microg of Tf-lipoplexes per microgram of plasmid. For intratumoral studies, CT26 (5 x 105 cells) in 50 microl of PBS were inoculated subcutaneously into the back of the mouse. Treatments began when tumor sizes reached 5-6 mm in diameter. Complexes were injected by a single intratumoral injection in a volume of 50 microl. Our in vitro results indicate that Tf-lipoplexes always mediate higher gene expression in colon (CT26) tumor cells, compared to plain-lipoplexes (without ligand) or naked plasmid. At the same time, CT26 tumor-bearing animals treated with Tf-lipoplexes containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice (p < 0.001), without signs of recurrence. High levels of IL-12 and IFN-gamma were detected in the sera of treated mice. Mice survival also improved considerably by treatment with this system, with a survival rate of 88%, at 23 days post-administration. In summary, in this study we have developed an efficient, targeted cationic lipid-based system for the treatment of colon tumors. The vector has the advantages of ease of preparation and economy, in comparison with commercial transfection reagents, as well as, the possibility of a large scale production.

  1. The 3'UTR 1188 A/C polymorphism in the interleukin-12p40 gene (IL-12B) is associated with lepromatous leprosy in the west of Mexico.

    PubMed

    Alvarado-Navarro, Anabell; Montoya-Buelna, Margarita; Muñoz-Valle, José Francisco; López-Roa, Rocio Ivette; Guillén-Vargas, Cecilia; Fafutis-Morris, Mary

    2008-06-30

    Leprosy is a chronic infectious disease caused by Mycobacterium leprae. IL-12 participates in the immune response against M. leprae by regulating T cell differentiation into the Th1-type response. Several single nucleotide polymorphisms have been identified in the IL-12 gene such as 3'UTR 1188 A/C polymorphism, which is associated with different diseases. However, the relationship of this polymorphism with the immune response in leprosy has not been explored. In this case-control study, we evaluated 44 patients with lepromatous leprosy (LL) and 51 healthy subjects (HS). We aimed to determine the relationship between 3'UTR 1188 A/C polymorphism of IL-12 p40, mRNA expression, and soluble IL-12 concentration in LL patients and HS. Genotype frequencies were 41% A/A, 36% A/C, and 23% C/C in LL patients, and 47% A/A, 49% A/C, and 4% C/C in HS (p<0.05). LL patients had a lower mRNA expression of IL-12 p40 gene, whereas HS had a higher expression level. Soluble IL-12 p40 concentration was higher in LL patients than in HS (p<0.05). IL-12 p70 concentration did not differ between groups, and IL-12 p40 concentration was not significantly correlated with mRNA expression in either group. These data suggest that IL-12 p40 3'UTR 1188 A/C polymorphism is associated with greater susceptibility to lepromatous leprosy in patients from western Mexico, independently of IL-12 p40 and p70 expression levels.

  2. Local, non-viral IL-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment.

    PubMed

    Janát-Amsbury, Margit Maria; Yockman, James W; Lee, Minhyung; Kern, Steven; Furgeson, Darin Y; Bikram, Malavosklish; Kim, Sung Wan

    2005-01-03

    Development of improved gene transfer methods is needed for gene therapy to achieve its clinical potential. The use of biocompatible polymeric gene carriers has shown effectiveness in overcoming the current problems associated with viral vectors in safety, immunogenicity and mutagenesis. Previous work has demonstrated that repeated, local, non-viral interleukin-12 (IL-12) gene delivery successfully slows down tumor progression, while improving immunogenicity. Combining IL-12 gene delivery with systemic paclitaxel (PCT) chemotherapy as a treatment for various subcutaneous mouse mammary carcinomas, we used PCT with either a biodegradable polymeric solubilizer, HySolv or Cremophor EL for systemic treatment and injected water soluble lipopolymer (WSLP)/plasmid-encoding IL-12 gene (p2CMVmIL-12) complexes local once every week. The amount of lung metastases being essential for survival as well as subcutaneous tumor volume were compared against untreated controls. We showed inhibition of tumor growth and decreased lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the PCT only treated groups. Compared to Cremophor, HySolv performed better alone or in combination with IL-12. Using polymeric vectors as gene carrier systems in combination with improved systemic therapies provide evidence for the efficacy and feasibility of polymer-based drug delivery systems. Especially local cytokine gene delivery showed augmentation of systemic chemotherapy while reducing the hosts risk for further systemic toxicity.

  3. A Large-Scale Genetic Association Study Confirms IL12B and Leads to the Identification of IL23R as Psoriasis-Risk Genes

    PubMed Central

    Cargill, Michele ; Schrodi, Steven J. ; Chang, Monica ; Garcia, Veronica E. ; Brandon, Rhonda ; Callis, Kristina P. ; Matsunami, Nori ; Ardlie, Kristin G. ; Civello, Daniel ; Catanese, Joseph J. ; Leong, Diane U. ; Panko, Jackie M. ; McAllister, Linda B. ; Hansen, Christopher B. ; Papenfuss, Jason ; Prescott, Stephen M. ; White, Thomas J. ; Leppert, Mark F. ; Krueger, Gerald G. ; Begovich, Ann B. 

    2007-01-01

    We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric single-nucleotide polymorphisms (SNPs) and found a highly significant association with an IL12B 3′-untranslated-region SNP (rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio [OR]common 0.64, combined P [Pcomb]=7.85×10-10). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of IL12B were resequenced in 96 individuals with psoriasis, and 30 additional IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (rs6887695) located ∼60 kb upstream of the IL12B coding region that exhibited association with psoriasis after adjustment for rs3212227. Together, these two SNPs mark a common IL12B risk haplotype (ORcommon 1.40, Pcomb=8.11×10-9) and a less frequent protective haplotype (ORcommon 0.58, Pcomb=5.65×10-12), which were statistically significant in all three studies. Since IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (IL12A and IL23A) and their receptors (IL12RB1, IL12RB2, and IL23R). Haplotype analyses identified two IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (ORcommon 1.44, Pcomb=3.13×10-6). Individuals homozygous for both the IL12B and the IL23R predisposing haplotypes have an increased risk of disease (ORcommon 1.66, Pcomb=1.33×10-8). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis pathogenesis. PMID:17236132

  4. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes.

    PubMed

    Cargill, Michele; Schrodi, Steven J; Chang, Monica; Garcia, Veronica E; Brandon, Rhonda; Callis, Kristina P; Matsunami, Nori; Ardlie, Kristin G; Civello, Daniel; Catanese, Joseph J; Leong, Diane U; Panko, Jackie M; McAllister, Linda B; Hansen, Christopher B; Papenfuss, Jason; Prescott, Stephen M; White, Thomas J; Leppert, Mark F; Krueger, Gerald G; Begovich, Ann B

    2007-02-01

    We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric single-nucleotide polymorphisms (SNPs) and found a highly significant association with an IL12B 3'-untranslated-region SNP (rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio [OR](common) 0.64, combined P [Pcomb]=7.85x10(-10)). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of IL12B were resequenced in 96 individuals with psoriasis, and 30 additional IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (rs6887695) located approximately 60 kb upstream of the IL12B coding region that exhibited association with psoriasis after adjustment for rs3212227. Together, these two SNPs mark a common IL12B risk haplotype (OR(common) 1.40, Pcomb=8.11x10(-9)) and a less frequent protective haplotype (OR(common) 0.58, Pcomb=5.65x10(-12)), which were statistically significant in all three studies. Since IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (IL12A and IL23A) and their receptors (IL12RB1, IL12RB2, and IL23R). Haplotype analyses identified two IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (OR(common) 1.44, Pcomb=3.13x10(-6)). Individuals homozygous for both the IL12B and the IL23R predisposing haplotypes have an increased risk of disease (OR(common) 1.66, Pcomb=1.33x10(-8)). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis

  5. Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

    PubMed

    Kato-Kogoe, Nahoko; Ohyama, Hideki; Okano, Soichiro; Yamanegi, Koji; Yamada, Naoko; Hata, Masaki; Nishiura, Hiroshi; Abiko, Yoshimitsu; Terada, Nobuyuki; Nakasho, Keiji

    2016-01-01

    Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.

  6. The Relationship between the Antitumor Effect of the IL-12 Gene Therapy and the Expression of Th1 Cytokines in an HPV16-Positive Murine Tumor Model

    PubMed Central

    García Paz, Flor; Madrid Marina, Vicente; Morales Ortega, Ausencio; Santander González, Abimelec; Peralta Zaragoza, Oscar; Burguete García, Ana; Torres Poveda, Kirvis; Moreno, José; Alcocer González, Juan; Hernandez Marquez, Eva; Bermúdez Morales, Victor

    2014-01-01

    Objective. The goal of the present study was to investigate the effect of IL-12 expressed in plasmid on the Th1 cytokine profile in an experimental HPV16-positive murine tumor model and the association with the IL-12's antitumor effect. Methods. Mice were injected with BMK-16/myc cells to establish HPV16-positive tumor and then pNGVL3-mIL-12 plasmid; pcDNA3 plasmid or PBS was injected directly into tumor site. The antitumor effect of the treatment was evaluated and the cytokines expression profile in each tumor tissue was analyzed. Results. Treatment with pNGVL3-mIL-12 plasmid had a significant antitumor effect, and a Th2-Th3-type cytokines prolife was detected in the murine tumor model with expression of the cytokines IL-10, IL-4, and TGF-β1. However, after the tumor was treated with three intratumoral injections of plasmid containing IL-12 cDNA, it showed a cytokine profile associated with Th1 with expression of IL-2, IL-12, and IFN-γ cytokines and reduced expression of IL-10, IL-4, and TGF-β1. Conclusions. The treatment with the IL-12 gene in the experimental HPV16-positive tumor model promoted the activation of the cellular immune response via expression of a Th1-type cytokine profile and was associated with the inhibition of tumor growth. Thus, IL-12 treatment represents a novel approach for gene therapy against cervical cancer. PMID:24808638

  7. Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

    PubMed

    Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

    2010-01-01

    The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

  8. A 1,100-year-old founder effect mutation in IL12B gene is responsible for Mendelian susceptibility to mycobacterial disease in Tunisian patients.

    PubMed

    Ben-Mustapha, Imen; Ben-Ali, Meriem; Mekki, Najla; Patin, Etienne; Harmant, Christine; Bouguila, Jihène; Elloumi-Zghal, Houda; Harbi, Abdelaziz; Béjaoui, Mohamed; Boughammoura, Lamia; Chemli, Jalel; Barbouche, Mohamed-Ridha

    2014-01-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder predisposing apparently healthy individuals to infections caused by weakly virulent mycobacteria such as bacille Calmette-Guerin (BCG), environmental mycobacteria, and poorly virulent Salmonella strains. IL-12p40 deficiency is the first reported human disease due to a cytokine gene defect and is one of the deficiencies that cause MSMD. Nine mutant alleles only have been identified in the IL12B gene, and three of them are recurrent mutations due to a founder effect in specific populations. IL-12p40 deficiency has been identified especially in countries where consanguinity is high and where BCG vaccination at birth is universal. We investigated, in such settings, the clinical, cellular, and molecular features of six IL-12p40-deficient Tunisian patients having the same mutation in IL12B gene (c.298_305del). We found that this mutation is inherited as a common founder mutation arousing ~1,100 years ago. This finding facilitates the development of a preventive approach by genetic counseling and prenatal diagnosis especially in affected families.

  9. Chemotherapy, IL-12 gene therapy and combined adjuvant therapy of HPV 16-associated MHC class I-proficient and -deficient tumours.

    PubMed

    Indrová, Marie; Bieblová, Jana; Jandlová, Tána; Vonka, Vladimír; Pajtasz-Piasecka, Elzbieta; Reinis, Milan

    2006-01-01

    Moderately immunogenic HPV 16-associated murine tumour cell line mimicking human HPV 16-associated neoplasms TC-1 (MHC class I(+)) and its variants, TC-1/P3C10 and TC-1/A9, with a marked down-regulation of MHC I molecules, were used to examine the effect of local interleukin 12 (IL-12) gene therapy for the treatment of early tumour transplants and minimal residual tumour disease obtained after cytoreductive chemotherapy (CMRTD). Experiments were designed to examine whether down-regulation of MHC class I molecules plays a role during chemotherapy and gene therapy of early tumour transplants. It was found that peritumoral administration of IL-12-producing tumour cell vaccines (single dose, day 8 after tumour cell administration) inhibited the growth of both TC-1 (MHC class I positive) tumours and their MHC class I-deficient variants. To investigate the antitumour effects in a clinically relevant setting, IL-12 gene therapy was utilised for the treatment of minimal residual tumour disease after cytoreductive chemotherapy. Intra-peritoneal treatment of tumour-bearing mice with ifosfamide derivative, CBM-4A, produced a significant tumour-inhibitory effect. This treatment was followed by peritumoral s.c. administration of genetically modified TC-1 (MHC class I positive) or MK16/I/IIIABC (MHC class I negative) vaccines producing IL-12 (single dose, day 7 after chemotherapy) or with recombinant interleukin 12 (rIL-12) in two cycles of 5 daily doses (days 8-19) after chemotherapy. This combined therapy significantly inhibited the growth of TC-1 and TC-1/A9 (MHC class I-) tumours. When the combined therapy of TC-1 (MHC class I positive) tumours was followed by peritumoral administration of bone marrow dendritic cell (BMDC) vaccines, the IL-12-mediated inhibitory effect was significantly boosted. In the next set of experiments, the impacts of chemotherapy and IL-12 adjuvant therapy on MHC class I surface expression were assessed. Chemotherapy and gene therapy of tumours led

  10. Serum interleukin (IL)-10 and IL-12 levels and IL28B gene polymorphisms: pretreatment prediction of treatment failure in chronic hepatitis C.

    PubMed

    Umemura, Takeji; Joshita, Satoru; Yoneda, Suguru; Katsuyama, Yoshihiko; Ichijo, Tetsuya; Matsumoto, Akihiro; Yoshizawa, Kaname; Ota, Masao; Tanaka, Eiji

    2011-01-01

    Both IL28B gene polymorphisms and serum levels of interleukin (IL)-10, IL-12p40 and IL-18 have been reported to affect the outcome of natural and pegylated interferon and ribavirin-treated HCV infection. To clarify their association and predictive value in treatment outcome of genotype 1 HCV-infected patients, we measured pretreatment serum IL-10, IL-12p40 and IL-18 levels using multiplex assays and determined IL28B gene polymorphisms (rs 8099917) in 52 cases with chronic hepatitis C. High baseline levels of IL-10 (P<0.001) and low levels of IL-12p40 (P<0.001) were significantly associated with a non-virological response (NVR) in our cohort. The IL28B polymorphism was tested and TT, TG or GG genotypes were found in 60%, 38% and 2% of patients, respectively, with corresponding NVR rates of 10%, 60% and 100% (P<0.001). Serum cytokine levels were significantly correlated with IL28B gene polymorphisms. When serum IL-10 levels were stratified at 5.0 pg/ml, NVR rates were 50% versus 0% (P=0.004) for the TT genotype and 87% versus 0% (P=0.001) for the TG or GG genotypes. Similarly, low IL-12p40 levels were associated with an NVR in patients with TG or GG genotypes (P=0.006). In multivariate analysis, high IL-10, low IL-12p40 and IL28B TG or GG genotypes were independently associated with an NVR. Serum IL-10 and IL-12p40 levels in combination with IL28B genotype, especially G-allele carriage, are strong predictive markers of an NVR to HCV treatment with pegylated interferon and ribavirin.

  11. Association of IL-12B gene rs6887695 polymorphism with hereditary susceptibility and clinical characterization of psoriasis vulgaris in the Chinese Han population.

    PubMed

    Wu, Yumei; Lu, Zhiyong; Chen, Yingwei; Xue, Feng; Chen, Xiaoying; Pan, Meng; Zheng, Jie

    2013-08-01

    We aimed to investigate the role of IL-12B gene polymorphism (rs6887695) in the disease susceptibility and clinical phenotypes of psoriasis vulgaris patients in the Chinese Han population. The genotype data of the IL-12B gene polymorphism (rs6887695) in 575 psoriasis patients and 1,403 normal controls were investigated using TaqMan technology. The Chi-square test was used to compare the genotype and allele frequency distribution among the groups. The genotypic and allelic frequencies of rs6887695 in the IL-12B gene between the cases and controls, as well as between the guttate and plaque psoriasis cases, were statistically significant (P genotype <0.01, P allele <0.01). However, the differences between the pediatric and adult onset psoriasis patients, between familial and sporadic cases, and between female and male cases were not statistically significant (P > 0.05). The genetic polymorphism of the IL-12B gene (rs6887695) may be associated with the psoriasis susceptibility in the Chinese Han population, especially for the plaque cases, but not associated with the age at onset, family history, or sex.

  12. Gene Electrotransfer of Plasmid-Encoding IL-12 Recruits the M1 Macrophages and Antigen-Presenting Cells Inducing the Eradication of Aggressive B16F10 Murine Melanoma

    PubMed Central

    Lampreht Tratar, Ursa; Loiacono, Luisa; Kamensek, Urska; Fazio, Vito Michele

    2017-01-01

    Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12. PMID:28596641

  13. Replication study of 10 genes showing evidence for association with multiple sclerosis: validation of TMEM39A, IL12B and CBLB [correction of CLBL] genes.

    PubMed

    Varadé, Jezabel; Comabella, Manuel; Ortiz, Miguel A; Arroyo, Rafael; Fernández, Oscar; Pinto-Medel, M Jesús; Fedetz, María; Izquierdo, Guillermo; Lucas, Miguel; Gómez, Carlos López; Rabasa, Antonio Catalá; Alcina, Antonio; Matesanz, Fuencisla; Alloza, Iraide; Antigüedad, Alfredo; García-Barcina, María; Otaegui, David; Olascoaga, Javier; Saiz, Albert; Blanco, Yolanda; Montalbán, Xavier; Vandenbroeck, Koen; Urcelay, Elena

    2012-07-01

    Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p(M-H)=0.001; OR(M-H) (95% CI)= 0.84 (0.75-0.93)], IL12B [rs6887695: p(M-H)=0.03; OR(M-H) (95% CI)= 1.09 (1.01-1.17)] and CBLB [rs9657904: p(M-H)=0.01; OR(M-H) (95% CI)= 0.89 (0.81-0.97)].

  14. Analysis of the IL-10, IL-12, and TNF-α Gene Polymorphisms in Patients With Vesicoureteral Reflux Among the Southeast Iranian Population

    PubMed Central

    Kordi Tamandani, Dor Mohammad; Naeimi, Nasim; Ghasemi, Ali; Baranzahi, Taybe; Sadeghi-Bojd, Simin

    2016-01-01

    Background Vesicoureteral reflux (VUR) is a common childhood disorder that is characterized by the abnormal movement of urine from the bladder into the ureters or kidneys. Objectives The aim of this study was to determine whether the genetic polymorphisms of the IL-10, IL-12, and TNF-α genes are involved in the development of VUR. Patients and Methods The tetra amplification mutation refractory system-polymerase chain reaction (Tetra-ARMS PCR) was applied to analyze the four polymorphic sites of the IL-10AG-1082, IL-10CA597, IL-12CA1188, and TNF308GA genes in 124 VUR children and 110 healthy controls. Results A significant, highly increased risk of VUR disease was found for the CA, AA, and combined genotypes of IL-10CA597 (OR = 5.2, 95% CL: 1.80 - 18.25; P = 0.0006, OR = 9.1, 95% CL: 1.11 - 122.75; P = 0.02, OR = 5.3, 95% CL: 1.82 - 18.61; P = 0.00052, respectively); the AG, GG, and AG + GG genotypes of IL-10AG-1082 (OR = 12.8, 95% CL; 2.9 - 113.9; P = 0.00003, OR = 12.62, 95% CL: 2.93 - 114.53; P = 0.00003, respectively); and the AA genotype of IL-12 (AA, OR = 0.19, 95% CL: 0.5 - 0.55; P = 0.0006). The frequency of the C allele in both IL-10CA and IL-12CA was greater in patients with VUR than in the healthy controls. No association was found between TNF308GA and the risk of VUR. Conclusions The results demonstrated significant associations between the IL-10 (AG-1089, IL-10CA) and IL-12 (AA) gene polymorphisms and a highly increased risk of VUR. PMID:27247930

  15. A GWAS follow-up study reveals the association of the IL12RB2 gene with systemic sclerosis in Caucasian populations

    PubMed Central

    Bossini-Castillo, Lara; Martin, Jose-Ezequiel; Broen, Jasper; Gorlova, Olga; Simeón, Carmen P.; Beretta, Lorenzo; Vonk, Madelon C.; Luis Callejas, Jose; Castellví, Ivan; Carreira, Patricia; José García-Hernández, Francisco; Fernández Castro, Mónica; Coenen, Marieke J.H.; Riemekasten, Gabriela; Witte, Torsten; Hunzelmann, Nicolas; Kreuter, Alexander; Distler, Jörg H.W.; Koeleman, Bobby P.; Voskuyl, Alexandre E.; Schuerwegh, Annemie J.; Palm, Øyvind; Hesselstrand, Roger; Nordin, Annika; Airó, Paolo; Lunardi, Claudio; Scorza, Raffaella; Shiels, Paul; van Laar, Jacob M.; Herrick, Ariane; Worthington, Jane; Denton, Christopher; Tan, Filemon K.; Arnett, Frank C.; Agarwal, Sandeep K.; Assassi, Shervin; Fonseca, Carmen; Mayes, Maureen D.; Radstake, Timothy R.D.J.; Martin, Javier

    2012-01-01

    A single-nucleotide polymorphism (SNP) at the IL12RB2 locus showed a suggestive association signal in a previously published genome-wide association study (GWAS) in systemic sclerosis (SSc). Aiming to reveal the possible implication of the IL12RB2 gene in SSc, we conducted a follow-up study of this locus in different Caucasian cohorts. We analyzed 10 GWAS-genotyped SNPs in the IL12RB2 region (2309 SSc patients and 5161 controls). We then selected three SNPs (rs3790567, rs3790566 and rs924080) based on their significance level in the GWAS, for follow-up in an independent European cohort comprising 3344 SSc and 3848 controls. The most-associated SNP (rs3790567) was further tested in an independent cohort comprising 597 SSc patients and 1139 controls from the USA. After conditional logistic regression analysis of the GWAS data, we selected rs3790567 [PMH= 1.92 × 10−5 odds ratio (OR) = 1.19] as the genetic variant with the firmest independent association observed in the analyzed GWAS peak of association. After the first follow-up phase, only the association of rs3790567 was consistent (PMH= 4.84 × 10−3 OR = 1.12). The second follow-up phase confirmed this finding (Pχ2 = 2.82 × 10−4 OR = 1.34). After performing overall pooled-analysis of all the cohorts included in the present study, the association found for the rs3790567 SNP in the IL12RB2 gene region reached GWAS-level significant association (PMH= 2.82 × 10−9 OR = 1.17). Our data clearly support the IL12RB2 genetic association with SSc, and suggest a relevant role of the interleukin 12 signaling pathway in SSc pathogenesis. PMID:22076442

  16. IL12 — EDRN Public Portal

    Cancer.gov

    The cytokine IL12, also known as IL12-p70, is the heterodimer formed by the protein products of the IL12A and IL12B genes. IL12A has a molecular weight of 35 kD, IL12B has a molecular weight of 40 kD, and together they are known as IL12-p70. From NCBI Gene: The cytokine (IL12) is a disulfide-linked heterodimer composed of the 35-kD subunit encoded by this gene, and a 40-kD subunit that is a member of the cytokine receptor family. This cytokine is required for the T-cell-independent induction of interferon (IFN)-gamma, and is important for the differentiation of both Th1 and Th2 cells. The responses of lymphocytes to this cytokine are mediated by the activator of transcription protein STAT4. Nitric oxide synthase 2A (NOS2A/NOS2) is found to be required for the signaling process of this cytokine in innate immunity. [provided by RefSeq, Jul 2008

  17. In situ cytokines (IL-4, IL-10, IL-12, IFN-γ) and chemokines (MCP-1, MIP-1α) gene expression in human Leishmania (Leishmania) Mexicana infection.

    PubMed

    Valencia-Pacheco, Guillermo; Loría-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Canché-Pool, Elsy B; Vargas-Gonzalez, Alberto; Melby, Peter C; Andrade-Narvaez, Fernando J

    2014-09-01

    Crucial to the defense against Leishmania is the ability of the host to mount a cell-mediated immune response capable of controlling and/or eliminating the parasite. The composition of the cell populations recruited in the early phase of the infection seems to be essential for defining the infection outcomes. The signals that initiate and regulate the early immune response and local accumulation of cell subsets in the skin are poorly understood. We previously studied the in situ expression of cytokine genes in patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. In the present study we examined in situ cytokine (IL-4, IL-10, IL-12, IFN-γ) and chemokine (MCP-1, MIP-1α) gene expression in L. (L.) mexicana active LCL lesions, and in the delayed type hypersensitivity (DTH) skin response to Leishmania antigen in subjects with healed lesion and subclinical infection. Data regarding cytokines were similar to previous studies in patients with active LCL. There were no significant differences in the profile of cytokine and chemokine gene expression in DTH from subjects with healed or subclinical infection. IL-12 gene expression detected in both groups was similar. High expression of MCP-1 was detected in all patients with active LCL. There was no difference in the level of MCP-1 expression between the healed lesion and the subclinical infection groups (p = 0.876). IL-12 and MCP-1 in the absence of IFN-γ might be playing a crucial role in infection outcomes at skin level.

  18. IL-12Rβ1 deficiency: mutation update and description of the IL12RB1 variation database

    PubMed Central

    van de Vosse, Esther; Haverkamp, Margje H.; Ramirez-Alejo, Noe; Martinez-Gallo, Mónica; Blancas-Galicia, Lizbeth; Metin, Ayşe; Garty, Ben Zion; Sun-Tan, Çağman; Broides, Arnon; de Paus, Roelof A.; Keskin, Özlem; Çağdaş, Deniz; Tezcan, Ilhan; Lopez-Ruzafa, Encarna; Aróstegui, Juan I.; Levy, Jacov; Espinosa-Rosales, Francisco J.; Sanal, Özden; Santos-Argumedo, Leopoldo; Casanova, Jean-Laurent; Boisson-Dupuis, Stephanie; van Dissel, Jaap T.; Bustamante, Jacinta

    2014-01-01

    IL-12Rβ1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL-12Rβ1 is a receptor chain of both the IL-12 and the IL-23 receptor and deficiency of IL-12Rβ1 thus abolishes both IL-12 and IL-23 signaling. IL-12Rβ1 deficiency is caused by bi-allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL-12Rβ1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL-12Rβ1 protein. In addition to disease-causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL-12Rβ1 and molecular genetics of human IL12RB1. PMID:23864330

  19. Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma.

    PubMed

    Xu, Yu; Hou, Jinxuan; Liu, Zhengchun; Yu, Haijun; Sun, Wenjie; Xiong, Jie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; Zhou, Yunfeng

    2011-04-11

    Gene therapy is a promising therapeutic approach for cancer. Targeted expression of desired therapeutic proteins within the tumor is the best approach to reduce toxicity and improve survival. This study is to establish a more effective and less toxic gene therapy of cancer. Combined gene therapy strategy with recombinant adenovirus expressing horseradish peroxidase (HRP) mediated by human telomerase reverse transcriptase (hTERT) promoter (AdhTERTHRP) and murine interleukin-12 (mIL-12) under the control of Cytomegalovirus (CMV) promoter (AdCMVmIL-12) was developed and evaluated against Lewis lung carcinoma (LLC) both in vivo and in vitro. The mechanism of action and systemic toxicities were also investigated. The combination of AdhTERTHRP/indole-3-acetic acid (IAA) treatment and AdCMVmIL-12 resulted in significant tumor growth inhibition and survival improvement compared with AdhTERTHRP/IAA alone (tumor volume, 427.4 ± 48.7 mm3 vs 581.9 ± 46.9 mm3, p = 0.005 on day 15; median overall survival (OS), 51 d vs 33 d) or AdCMVmIL-12 alone (tumor volume, 362.2 ± 33.8 mm3 vs 494.4 ± 70.2 mm3, p = 0.046 on day 12; median OS, 51 d vs 36 d). The combination treatment stimulated more CD4+ and CD8+ T lymphocyte infiltration in tumors, compared with either AdCMVmIL-12 alone (1.3-fold increase for CD4+ T cells and 1.2-fold increase for CD8+ T cells, P < 0.01) or AdhTERTHRP alone (2.1-fold increase for CD4+ T cells and 2.2-fold increase for CD8+ T cells, P < 0.01). The apoptotic cells in combination group were significantly increased in comparison with AdCMVmIL-12 alone group (2.8-fold increase, P < 0.01) or AdhTERTHRP alone group (1.6-fold increase, P < 0.01). No significant systematic toxicities were observed. Combination gene therapy with AdhTERTHRP/IAA and AdCMVmIL-12 could significantly inhibit tumor growth and improve host survival in LLC model, without significant systemic adverse effects.

  20. Common polymorphisms in interleukin genes (IL4, IL6, IL8 and IL12) are not associated with alcoholic liver disease or alcoholism in Spanish men.

    PubMed

    Marcos, Miguel; Pastor, Isabel; González-Sarmiento, Rogelio; Laso, Francisco-Javier

    2009-03-01

    Preliminary data suggest that polymorphisms in cytokine genes may be involved in the genetic predisposition to alcoholic liver cirrhosis or alcohol use disorders. We thus analyze the association between these diseases and the following polymorphisms: -33T>C IL4, -174 G>C IL6, -251 T>A IL8 and 1188 A>C IL12B. 258 male alcoholics (161 without liver disease and 97 with liver cirrhosis) and 101 healthy controls were genotyped for the above mentioned polymorphisms. We examined the relationship between genotype and allele frequencies and the presence of disease, as well as the correlation with combinations of putative pro-inflammatory genotypes. Haplotypes were inferred using the expectation-maximization algorithm and haplotype frequencies were compared. We found no statistically significant association between any of these polymorphisms or the combinations of pro-inflammatory polymorphisms and the risk of alcoholic liver cirrhosis or alcohol abuse or dependence. Haplotype analysis of the IL4 and IL12B polymorphisms did not show any statistical relationship either. Our results do not support the hypothesis that the analyzed polymorphisms confer differences in alcoholic liver cirrhosis or alcohol use disorders susceptibility.

  1. Association Study of Genes Controlling IL-12-dependent IFN-γ Immunity: STAT4 Alleles Increase Risk of Pulmonary Tuberculosis in Morocco

    PubMed Central

    Sabri, Ayoub; Grant, Audrey V.; Cosker, Kristel; El Azbaoui, Safa; Abid, Ahmed; Abderrahmani Rhorfi, Ismail; Souhi, Hicham; Janah, Hicham; Alaoui-Tahiri, Kebir; Gharbaoui, Yasser; Benkirane, Majid; Orlova, Marianna; Boland, Anne; Deswarte, Caroline; Migaud, Melanie; Bustamante, Jacinta; Schurr, Erwin; Boisson-Dupuis, Stephanie; Casanova, Jean-Laurent; Abel, Laurent; El Baghdadi, Jamila

    2014-01-01

    Background. Only a minority of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis. Genetic epidemiological evidence suggests that pulmonary tuberculosis has a strong human genetic component. Previous genetic findings in Mendelian predisposition to more severe mycobacterial infections, including by M. tuberculosis, underlined the importance of the interleukin 12 (IL-12)/interferon γ (IFN-γ) circuit in antimycobacterial immunity. Methods. We conducted an association study in Morocco between pulmonary tuberculosis and a panel of single-nucleotide polymorphisms (SNPs) covering 14 core IL-12/IFN-γ circuit genes. The analyses were performed in a discovery family-based sample followed by replication in a case-control population. Results. Out of 228 SNPs tested in the family-based sample, 6 STAT4 SNPs were associated with pulmonary tuberculosis (P = .0013–.01). We replicated the same direction of association for 1 cluster of 3 SNPs encompassing the promoter region of STAT4. In the combined sample, the association was stronger among younger subjects (pulmonary tuberculosis onset <25 years) with an odds ratio of developing pulmonary tuberculosis at rs897200 for GG vs AG/AA subjects of 1.47 (1.06–2.04). Previous functional experiments showed that the G allele of rs897200 was associated with lower STAT4 expression. Conclusions. Our present findings in a Moroccan population support an association of pulmonary tuberculosis with STAT4 promoter-region polymorphisms that may impact STAT4 expression. PMID:24610875

  2. Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds.

    PubMed

    Sailaja, M; Tarakeswari, M; Sujatha, M

    2008-09-01

    The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l(-1) thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding beta-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 microm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l(-1) BA and hygromycin (20, 40 and 60 mg l(-1)) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T0 and T1 plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.

  3. Gene gun-mediated DNA vaccination enhances antigen-specific immunotherapy at a late preclinical stage of type 1 diabetes in nonobese diabetic mice

    PubMed Central

    Goudy, Kevin S.; Wang, Bo; Tisch, Roland

    2008-01-01

    Type 1 diabetes (T1D) is characterized by the T cell mediated destruction of the insulin producing β cells. Antigen-specific immunotherapies are used to selectively tolerize β cell-specific pathogenic T cells either directly, or indirectly through the induction of immunoregulatory T cells. A key concern of antigen-specific immunotherapy is exacerbating autoimmunity. We compared the T cell reactivity and efficacy induced by plasmid DNA (pDNA) encoding glutamic acid decarboxylase 65 (GAD65) administered via intramuscular versus gene gun vaccination in NOD mice at a late preclinical stage of T1D. Whereas intramuscular injection of pGAD65 promoted a predominant type 1 CD4+ T cell response and failed to suppress ongoing β cell autoimmunity, gene gun vaccination preferentially induced IL-4 secreting CD4+ T cells and significantly delayed the onset of diabetes. These findings demonstrate that gene gun delivery of autoantigen-encoding pDNA preferentially elicits immunoregulatory T cells and offers a safe, effective mode of pDNA vaccination for the treatment of T1D and other autoimmune diseases. PMID:18675592

  4. Gene expression of subunits of the IL-12 family cytokines in moDCs derived in vitro from the cord blood of children of healthy and allergic mothers.

    PubMed

    Hrdý, J; Novotná, O; Kocourková, I; Prokešová, L

    2014-01-01

    The incidence of allergic diseases is steadily increasing an urgent need to clarify the immunologic processes which occur early in life and signal an increased risk of possible future allergy development. The ratio and maturation state of DCs together with the cytokine environment are important in directing and modulating immune responses. The maturation state (presence of CD83) of cord blood monocyte-derived dendritic cells (moDCs) of 52 children of healthy mothers and 58 children of allergic mothers was estimated by flow cytometry. The capacity of moDCs to express genes for subunits of IL-12 family cytokines was monitored using real-time PCR and protein secretion in cell culture supernatants by ELISA. The percentage of CD83+ moDCs was significantly higher in the allergic group after LPS stimulation (43.11 ± 4.41) in comparison to the healthy group (24.85 ± 3.37). Significantly higher gene expression of subunits of IL-12 family members was observed in moDCs of children of allergic mothers, in comparison with children of healthy mothers. The differences were evident mainly after LPS stimulation of moDCs (healthy group: p19: 3.05 ± 1.24; p28: 14.8 ± 6.8; p35: 1.8 ± 0.6; p40: 8.0 ± 3.5; EBI3: 3.0 ± 1.2; allergic group: p19: 6.1 ± 2.7; p28: 61.4 ± 22.2; p35: 14.9 ± 6.5; p40: 36.4 ± 18.8; EBI3: 11.3 ± 3.2), with the exception of p28, whose expression was significantly higher in the allergic group even without stimulation (healthy group: 0.28 ± 0.12, allergic group: 0.87 ± 0.62). No significant difference between the healthy and allergic groups was found at the protein level. The observation of both increased presence of cell surface activation marker on moDCs and higher IL-12 family gene expression in LPS-stimulated moDCs of children of allergic mothers indicates a higher reactivity of these cells.

  5. Inherited IL-12p40 Deficiency

    PubMed Central

    Prando, Carolina; Samarina, Arina; Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Cobat, Aurelie; Picard, Capucine; AlSum, Zobaida; Al-Jumaah, Suliman; Al-Hajjar, Sami; Frayha, Husn; Al-Mousa, Hamoud; Ben-Mustapha, Imen; Adimi, Parisa; Feinberg, Jacqueline; de Suremain, Maylis; Jannière, Lucile; Filipe-Santos, Orchidée; Mansouri, Nahal; Stephan, Jean-Louis; Nallusamy, Revathy; Kumararatne, Dinakantha S.; Bloorsaz, Mohamad Reza; Ben-Ali, Meriem; Elloumi-Zghal, Houda; Chemli, Jalel; Bouguila, Jihene; Bejaoui, Mohamed; Alaki, Emadia; AlFawaz, Tariq S.; Al Idrissi, Eman; ElGhazali, Gehad; Pollard, Andrew J.; Murugasu, Belinda; Wah Lee, Bee; Halwani, Rabih; Al-Zahrani, Mohammed; Al Shehri, Mohammed A.; Al-Zahrani, Mofareh; Bin-Hussain, Ibrahim; Mahdaviani, Seyed Alireza; Parvaneh, Nima; Abel, Laurent; Mansouri, Davood; Barbouche, Ridha; Al-Muhsen, Saleh

    2013-01-01

    Abstract Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is a rare genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunologic, and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia, and Tunisia). There are only 9 different mutant alleles of the IL12B gene: 2 small insertions, 3 small deletions, 2 splice site mutations, and 1 large deletion, each causing a frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacille Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (n = 3), nocardiosis (n = 2), and klebsiellosis (n = 1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor β1 (IL-12Rβ1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients

  6. Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study.

    PubMed

    Salvadori, Claudia; Svara, Tanja; Rocchigiani, Guido; Millanta, Francesca; Pavlin, Darja; Cemazar, Maja; Lampreht Tratar, Ursa; Sersa, Gregor; Tozon, Natasa; Poli, Alessandro

    2017-09-01

    The study was aimed to characterize tumor response after combined treatment employing electrochemotherapy with IL-12 gene electrotransfer in dogs with spontaneous mast cell tumors (MCT). Eleven dogs with eleven MCTs were included in the study. Histological changes were investigated in biopsy specimens collected before the treatment (T0), and 4 (T1) and 8 weeks (T2) later. Cellular infiltrates were characterized immunohistochemically by using anti CD3, CD20, Foxp3 (Treg), CD68 and anti MHC-class II antibodies. Proliferation and anti-apoptotic activity of neoplastic cells were assessed using anti Ki-67 and Bcl-2 antibodies. Angiogenetic processes were investigated immunohistochemically by using anti Factor VIII and anti CD31 antibodies and micro vessel density quantification. Histopathological examination of samples at T0 confirmed the diagnosis and the presence of scanty infiltrates consisted mainly of T-lymphocytes and macrophages. At T1 and T2 neoplastic cells were drastically reduced in 7/11 cases, small clusters of neoplastic cells were detected in 3/11 cases and 1/11 cases neoplastic cells were still evident. Proliferation activity of neoplastic cells was significantly reduced at T1 and T2 and expression of anti-apoptotic protein at T1. Microvessel density was drastically reduced in all samples after treatment. The number of T-lymphocytes increased at T1, although not significant, while Treg were significant higher at T1 and macrophages at T2. The combined electrochemotherapy and IL-12 gene electrotransfer effectively induced a cellular response against neoplastic cells characterized mainly by the recruitment of T-lymphocytes and macrophages and a fibrotic proliferation with reduction of microvessels.

  7. IL12Rβ1ΔTM Is a Secreted Product of il12rb1 That Promotes Control of Extrapulmonary Tuberculosis

    PubMed Central

    Ray, Aurelie A.; Fountain, Jeffrey J.; Miller, Halli E.; Cooper, Andrea M.

    2014-01-01

    IL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective TH1cells. Recently, we observed that il12rb1—the mouse homolog of IL12RB1—is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rβ1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in TH1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating TH cells response to IL-12. PMID:25404030

  8. Contribution of IL-12A and IL-12B polymorphisms to Chlamydia trachomatis-specific cell-mediated immune responses.

    PubMed

    Öhman, H; Natividad, A; Bailey, R; Ragoussis, J; Johnson, L-L; Tiitinen, A; Halttunen, M; Paavonen, J; Surcel, H-M

    2015-03-01

    Inherited variance in the IL-12B gene is associated with susceptibility to Chlamydia trachomatis-induced tubal factor infertility and disease severity. In this study, our aim was to discover how polymorphisms in IL-12-coding genes influence C. trachomatis-induced immune responses and IL-12 production. The study population consisted of 240 women. IL-12A and IL-12B single nucleotide polymorphisms (SNPs) were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We studied lymphocyte proliferative (LP) responses to C. trachomatis strains E and F elementary bodies (EBs) and recombinant chlamydial heat-shock protein 60 (CHSP60) antigen. IL-12p40 and IL-12p70 levels were measured using the BD Flex Set method. We found a statistically significant association between the C. trachomatis EB antigen-specific LP response and the rs2853694 SNP (P = 0.02). Our study demonstrates that the IL-12 cytokine family is involved in C. trachomatis-specific immune responses. Moreover, C. trachomatis-induced IL-12 production and the IL-12B rs2853694 SNP partially explain individual variation in the C. trachomatis LP response.

  9. Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif

    PubMed Central

    Leutenegger, Christian M.; Boretti, Felicitas S.; Mislin, Caroline N.; Flynn, J. Norman; Schroff, Matthias; Habel, Andre; Junghans, Claas; Koenig-Merediz, Sven A.; Sigrist, Brigitte; Aubert, Andre; Pedersen, Niels C.; Wittig, Burghardt; Lutz, Hans

    2000-01-01

    Four groups of cats, each containing four animals, were immunized at 0, 3, and 6 weeks with minimalistic immunogenic defined gene expression vector (MIDGE) vaccines containing the gene(s) for feline immunodeficiency virus (FIV) gp140, FIV gp140 and feline interleukin-12 (IL-12), FIV gp140 and feline IL-16, or FIV gp140 and a CpG motif. MIDGEs were coated onto gold beads and injected intradermally with a gene gun. A fifth group of four cats were immunized in an identical manner but with blank gold beads. All cats were challenge exposed to virulent FIV 4 weeks following the final immunization, and the course of infection was monitored. The two groups of cats immunized with the FIV gp140 gene alone or with blank gold particles became highly viremic and seroconverted as early as 4 weeks after infection. In contrast, three of four cats immunized with FIV gp140 in combination with feline IL-12 failed to become viremic or seropositive, as has been shown elsewhere (F. S. Boretti, C. M. Leutenegger, C. Mislin, et al., AIDS 14:1749–1757, 2000). Here we show the effect of IL-12 when used as an adjuvant on the viral RNA and DNA load and on the cytokine profile. In addition, the two groups of cats immunized either with gp140 and IL-16 or with gp140 and the CpG had greatly reduced viremia. Protection correlated weakly with cytotoxic T-lymphocyte activity and increased cytokine transcription of IL-12, gamma interferon, and IL-10 by peripheral blood mononuclear cells in the postchallenge period. This study extends the data on IL-12 and provides new results on CpG motifs and IL-16 used as adjuvants in the FIV cat model. PMID:11044089

  10. Rock bream (Oplegnathus fasciatus) IL-12p40: identification, expression, and effect on bacterial infection.

    PubMed

    Zhang, Lu; Zhang, Bao-Cun; Hu, Yong-Hua

    2014-08-01

    IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection.

  11. LincRNA-Cox2 modulates TNF-α-induced transcription of Il12b gene in intestinal epithelial cells through regulation of Mi-2/NuRD-mediated epigenetic histone modifications.

    PubMed

    Tong, Qiang; Gong, Ai-Yu; Zhang, Xin-Tian; Lin, Chengchi; Ma, Shibin; Chen, Jing; Hu, Guoku; Chen, Xian-Ming

    2016-03-01

    Long intergenic noncoding RNAs (lincRNAs) can regulate the transcription of inflammatory genes and thus may represent a new group of inflammatory mediators with a potential pathogenic role in inflammatory diseases. Here, our genome-wide transcriptomic data show that TNF-α stimulation caused up-regulation of 171 lincRNAs and down-regulation of 196 lincRNAs in murine intestinal epithelial cells in culture. One of the up-regulated lincRNAs, lincRNA-Cox2, is an early-responsive lincRNA induced by TNF-α through activation of the NF-ĸB signaling pathway. Knockdown of lincRNA-Cox2 resulted in reprogramming of the gene expression profile in intestinal epithelial cells in response to TNF-α stimulation. Specifically, lincRNA-Cox2 silencing significantly (P < 0.05) enhanced the transcription of Il12b, a secondary late-responsive gene induced by TNF-α. Mechanistically, lincRNA-Cox2 promoted the recruitment of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) repressor complex to the Il12b promoter region. Recruitment of the Mi-2/NuRD complex was associated with decreased H3K27 acetylation and increased H3K27 dimethylation at the Il12b promoter region, which might contribute to Il12b trans-suppression by lincRNA-Cox2. Thus, our data demonstrate a novel mechanism of epigenetic modulation by lincRNA-Cox2 on Il12b transcription, supporting an important role for lincRNAs in the regulation of intestinal epithelial inflammatory responses.

  12. IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes control of extrapulmonary tuberculosis.

    PubMed

    Ray, Aurelie A; Fountain, Jeffrey J; Miller, Halli E; Cooper, Andrea M; Robinson, Richard T

    2015-02-01

    IL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective T(H)1 cells. Recently, we observed that il12rb1—the mouse homolog of IL12RB1—is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rβ1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in TH1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating T(H) cells response to IL-12.

  13. The potential of interleukin 12 receptor beta 2 (IL12RB2) and tumor necrosis factor receptor superfamily member 8 (TNFRSF8) gene as diagnostic biomarkers of oral lichen planus (OLP).

    PubMed

    Jeon, Seung-Ho; Jeon, Eun-Hyoung; Lee, Jin-Yong; Kim, Yeon-Sun; Yoon, Hye-Jung; Hong, Sam-Pyo; Lee, Jong-Ho

    2015-01-01

    This study evaluated the potential of interleukin 12 receptor beta 2 and tumor necrosis factor receptor superfamily member 8 as diagnostic biomarkers of oral lichen planus (OLP). The mRNA expression of IL12RB2 and TNFRSF8 in FFPE OLP samples (OLP group, n = 38) were investigated with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis and compared to those of chronic non-specific mucositis (Non-OLP group, n = 25) and normal mucosa (Normal group, n = 18). Predictive modeling of the expression of IL12RB2 and TNFRSF8 was constructed using support vector machine (SVM), random forest (RF), linear discriminant analysis (LDA), neural network (NN) and naive Bayes (NB) methods. Normalized expression of IL12RB2 in the OLP group (3.78 ± 1.67) was significantly higher than the Normal group (1.97 ± 1.12), but lower than the Non-OLP group (6.86 ± 1.67). TNFRSF8 gene expression in the OLP group (7.46 ± 1.51) was significantly higher than the Normal group (2.90 ± 1.61), but no significant difference was found between the OLP and Non-OLP groups. The ratio of IL12RB2/TNFRSF8 in the OLP group (0.52 ± 0.23) was significantly lower than the Normal group (0.74 ± 0.39) and the Non-OLP group (1.07 ± 0.38). In the predictive modeling, the area under receiver operating characteristic (ROC) curves (AUC) ranged from 0.83-0.92 and their accuracy was higher than 0.75 in all methods. The IL12RB2/TNFRSF8 ratio can be a useful diagnostic tool for OLP.

  14. Infectious diseases, autoimmunity and midline defect in a patient with a novel bi-allelic mutation in IL12RB1 gene.

    PubMed

    Göktürk, Bahar; Reisli, İsmail; Çalışkan, Ümran; Oleaga-Quintas, Carmen; Deswarte, Caroline; Turul-Özgür, Tuba; Burgucu, Durmuş; Migaud, Melanie; Casanova, Jean-Laurent; Picard, Capucine; Bustamante, Jacinta

    2016-01-01

    Clinical disease caused by weakly pathogenic mycobacterial species, which is known as Mendelian susceptibility to mycobacterial disease (MSMD), is a rare entity. IFN-γ and IL-17 production are defective due to insufficient response to IL-2 and IL-23 in IL-12Rβ1 deficiency; so this also causes tendency to intracellular microorganisms and candidal diseases. Here, we present a patient who suffers IL-12Rβ1 deficiency caused by a novel bi-allelic mutation with recurrent salmonellosis, mycobacterial, fungal infections and remained asymptomatic during 13 months of follow-up after hIFN-γ treatment. In addition she had hemolytic anemia and midline defects like cleft lip and palate which have not been reported in a patient with MSMD in the literature prior to this case report. In conclusion, diagnosis of MSMD should be kept in mind in patients with recurrent salmonellosis, mycobacterial and fungal infections especially in countries with a high consanguinity rate.

  15. Optimized expression and specific activity of IL-12 by directed molecular evolution

    PubMed Central

    Leong, Steven R.; Chang, Jean C. C.; Ong, Randal; Dawes, Glenn; Stemmer, Willem P. C.; Punnonen, Juha

    2003-01-01

    DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration. PMID:12529500

  16. Human intestinal mucosa-associated Lactobacillus and Bifidobacterium strains with probiotic properties modulate IL-10, IL-6 and IL-12 gene expression in THP-1 cells.

    PubMed

    Čitar, M; Hacin, B; Tompa, G; Štempelj, M; Rogelj, I; Dolinšek, J; Narat, M; Matijašić, B Bogovič

    2015-01-01

    Lactobacilli and bifidobacteria are considered one of the permanent genera of the physiological human intestinal microbiota and represent an enormous pool of potential probiotic candidates. Approximately 450 isolates of presumptive Lactobacillus or Bifidobacterium strains were obtained from bioptic samples of colonic and ileal mucosa from 15 adolescents aged 12 to 18 years. On the basis of randomly amplified polymorphic DNA (RAPD)-PCR analysis, 20 strains were selected for further taxonomic classification and characterisation, as well as assessment of probiotic properties and safety. Importantly, selected strains showed the capability of colonising different parts of the intestine. The most frequently isolated species was Lactobacillus paracasei followed by Lactobacillus fermentum. The majority of isolates were susceptible to antimicrobials of human and veterinary importance, however, tetracycline and/or erythromycin resistance was observed in Lactobacillus plantarum and L. fermentum strains. Thirteen strains were able to ferment more than 19 different carbon sources and three out of five tested strains exerted antagonistic activity against several different indicator strains. Two Lactobacillus isolates (L. paracasei L350 and L. fermentum L930 bb) and one Bifidobacterium isolate (Bifidobacterium animalis subsp. animalis IM386) fulfilled in vitro selection criteria for probiotic strains and exhibited strong downregulation of pro-inflammatory cytokines IL-6 and IL-12 and upregulation of anti-inflammatory IL-10. The selected strains represent suitable candidates for further studies regarding their positive influence on host health and could play an important role in ameliorating the symptoms of inflammatory bowel diseases.

  17. Target-mediated disposition model describing the dynamics of IL12 and IFNγ after administration of a mifepristone-inducible adenoviral vector for IL-12 expression in mice.

    PubMed

    Parra-Guillen, Zinnia Patricia; Janda, Alvaro; Alzuguren, Pilar; Berraondo, Pedro; Hernandez-Alcoceba, Ruben; Troconiz, Iñaki F

    2013-01-01

    Interleukin-12 (IL12) is a cytokine with potential applications in the treatment of cancer given the potent immune response that it triggers, in part due to its ability to stimulate expression of interferon-γ (IFNγ). To avoid the toxicity associated with systemic exposure to IL12, a high-capacity adenoviral vector carrying a liver-specific, mifepristone-inducible IL12 expression system (HC-Ad/RUmIL12) has been developed. However, the maintenance of IL12 expression at therapeutic levels is compromised by the inhibitory effect of IFNγ on inducible systems. The aim of this work is to develop a semi-mechanistic model to characterize the relationship between IL12 and IFNγ in wild-type and knock-out mice for the IFNγ receptor treated with HC-Ad/RUmIL12 under different dosing regimens in order to better understand the key mechanisms controlling the system. Rapid binding was considered to account for target-mediated disposition exhibited by both cytokines (equilibrium dissociation constant were 18 and 2.28 pM for IL12 and IFNγ, respectively). The final model included: (1) IFNγ receptor turnover, (2) irreversible free cytokine elimination from the serum compartment, (3) internalization of the IL12 receptor complex, (4) IL12 expression upregulated by the co-administration of the adenoviral vector and mifepristone and downregulated by the IFNγ receptor, and (5) synthesis of IFNγ controlled by the relative increments in the bound IL12. In conclusion, a model simultaneously describing the kinetics of IL12 and IFNγ in the context of gene therapy was developed and validated with additional data. The model was applied to design an experimental dosing protocol intended to maintain sustained therapeutic IL12 levels.

  18. IL-12 family cytokines: immunological playmakers.

    PubMed

    Vignali, Dario A A; Kuchroo, Vijay K

    2012-07-19

    The interleukin 12 (IL-12) family is unique in having the only heterodimeric cytokines, including IL-12, IL-23, IL-27 and IL-35. This feature endows these cytokines with a unique set of connections and functional interactions not shared by other cytokine families. Despite sharing many structural features and molecular partners, cytokines of the IL-12 family mediate surprisingly diverse functional effects. Here we discuss the unique and unusual structural and functional characteristics of this cytokine family. We outline how cells might interpret seemingly similar cytokine signals to give rise to the diverse functional outcomes that characterize this cytokine family. We also discuss the therapeutic implications of this complexity.

  19. IL-12Rβ2 is essential for the development of experimental cerebral malaria.

    PubMed

    Fauconnier, Mathilde; Palomo, Jennifer; Bourigault, Marie-Laure; Meme, Sandra; Szeremeta, Frédéric; Beloeil, Jean-Claude; Danneels, Adeline; Charron, Sabine; Rihet, Pascal; Ryffel, Bernhard; Quesniaux, Valérie F J

    2012-02-15

    A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rβ2 in ECM development. C57BL/6 mice deficient for IL-12Rβ2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rβ2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rβ2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rβ2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rβ2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rβ2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.

  20. IL-12 protects from psoriasiform skin inflammation

    PubMed Central

    Kulig, Paulina; Musiol, Stephanie; Freiberger, Sandra Nicole; Schreiner, Bettina; Gyülveszi, Gabor; Russo, Giancarlo; Pantelyushin, Stanislav; Kishihara, Kenji; Alessandrini, Francesca; Kündig, Thomas; Sallusto, Federica; Hofbauer, Günther F.L.; Haak, Stefan; Becher, Burkhard

    2016-01-01

    Neutralization of the common p40-subunit of IL-12/23 in psoriasis patients has led to a breakthrough in the management of moderate to severe disease. Aside from neutralizing IL-23, which is thought to be responsible for the curative effect, anti-p40 therapy also interferes with IL-12 signalling and type 1 immunity. Here we dissect the individual contribution of these two cytokines to the formation of psoriatic lesions and understand the effect of therapeutic co-targeting of IL-12 and IL-23 in psoriasis. Using a preclinical model for psoriatic plaque formation we show that IL-12, in contrast to IL-23, has a regulatory function by restraining the invasion of an IL-17-committed γδT (γδT17) cell subset. We discover that IL-12 receptor signalling in keratinocytes initiates a protective transcriptional programme that limits skin inflammation, suggesting that collateral targeting of IL-12 by anti-p40 monoclonal antibodies is counterproductive in the therapy of psoriasis. PMID:27892456

  1. CD38 gene-modified dendritic cells inhibit murine asthma development by increasing IL-12 production and promoting Th1 cell differentiation.

    PubMed

    Wang, Jiaoli; Zhu, Weiguo; Chen, Yinghu; Lin, Zhendong; Ma, Shenglin

    2016-11-01

    Predominant T helper (Th)2 and impaired Th1 cell polarization has a crucial role in the development of asthma. Cluster of differentiation (CD)38 is associated with the increased release of interleukin (IL)‑12 from dendritic cells (DCs) and DC‑induced Th1 cell polarization. However, whether CD38 expression affects DC function in asthma development remains unknown. In the current study, adenoviruses were constructed containing the murine CD38 gene. Overexpression of CD38 protein level in DCs induced from bone‑marrow derived DCs (BMDCs) by recombinant mouse granulocyte macrophage colony‑stimulating factor and IL‑4 was achieved through 24 h adenovirus infection. The results demonstrated that BMDCs with CD38 overexpression exhibited no phenotypic change; however, following stimulation with lipopolysaccharide (LPS), maturation and IL‑12 secretion were increased. In addition, CD38‑overexpressing BMDCs stimulated with LPS exhibited more effective Th1 cell differentiation. Mice that were administered CD38‑overexpressing BMDCs exhibited milder symptoms of asthma. Furthermore, decreased IL‑4, IL‑5 and IL‑13 levels were detected in bronchoalveolar lavage fluid (BALF), reduced immunoglobulin E levels were measured in the sera, and increased interferon‑γ was detected in BALF from the recipients of CD38‑overexpressing BMDCs. Increased phosphorylated‑p38 expression was also detected in LPS-stimulated CD38-overexpressing BMDCs, whereas pretreatment with a p38‑specific inhibitor was able to abolish the effects of LPS stimulation and CD38 overexpression on IL‑12 release and Th1 cell differentiation in BMDCs. These results suggested that CD38 may be involved in the DC function of alleviating asthma via restoration of the Th1/Th2 balance, thus providing a novel strategy for asthma therapy.

  2. IL-12 Family Cytokines: General Characteristics, Pathogenic Microorganisms, Receptors, and Signalling Pathways.

    PubMed

    Behzadi, Payam; Behzadi, Elham; Ranjbar, Reza

    2016-03-01

    Among a wide range of cytokines, the Interleukin 12 (IL-12) family has its unique structural, functional, and immunological characteristics that have made this family as important immunological playmakers. Because of the importance of IL-12 heterodimeric cytokines in microbial infections, autoimmune diseases, and cancers, the authors of this literature discuss about the general characteristics of IL-12 family members, the interactions between IL-12 cytokines and pathogenic microorganisms, the interleukins receptors and their strategies for selecting different signalling pathways. IL-12 and IL-23 are similar in p40 subunits and both are involved in proinflammatory responses while, IL-27 and IL-35 contribute to anti-inflammatory activities; however, IL-27 is also involved in pro-inflammatory responses. There are some similarities and dissimilarities among IL-12 family members which make them a unique bridge between innate and adaptive immune systems. The bioactivities of IL-12 family indicate a brilliant promise for their applications in different fields of medicine. The members of IL-12 family are candidate for several therapeutics including gene therapy, cancer therapy, tumour therapy, and vaccination. To have an accurate diagnostic technique and definite treatment regarding to infectious diseases, the playmakers of IL-12 family as effective criteria together with microarray technology are the best choices for current and future applications.

  3. Rafting with the IL-12 receptor.

    PubMed

    McVicar, Daniel W

    2008-04-15

    In this issue of Blood, Kondadasula and colleagues provide important clues for understanding successful cancer immunotherapy by demonstrating an intriguing mechanism through which CD16 synergizes with IL-12 to induce interferon-gamma (IFN-gamma) production by NK cells.

  4. Role of the IL-12/IL-35 balance in Sjögren's syndrome.

    PubMed

    Fogel, Olivier; Rivière, Elodie; Seror, Raphaèle; Nocturne, Gaetane; Boudaoud, Saida; Ly, Bineta; Gottenberg, Jacques-Eric; Le Guern, Véronique; Dubost, Jean-Jacques; Nititham, Joanne; Taylor, Kimberly E; Chanson, Philippe; Dieudé, Philippe; Criswell, Lindsey A; Jagla, Bernd; Thai, Alice; Mingueneau, Michael; Mariette, Xavier; Miceli-Richard, Corinne

    2017-09-12

    An interferon (IFN) signature is involved in the pathogenesis of primary Sjögren's syndrome (pSS), but whether the signature is type 1 or 2 remains controversial. Mouse models and genetic studies suggested the involvement of T helper 1 and type 2 IFN pathways. Likewise, polymorphisms of interleukin 12A gene (IL-12A), which encodes for IL-12p35, have been associated with pSS. IL-12p35 subunit is shared by 2 heterodimers, IL-12 and IL-35. To confirm the genetic association of IL-12A polymorphism and pSS and elucidate the involvement of the IL-12/IL-35 balance in pSS by functional studies. The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French controls. Functional studies were performed on sorted monocytes, stimulated or not. IL-12A mRNA and IL-12 and IL-35 protein levels were assessed by qRT-PCR and by ELISA and a multiplex kit for IL-35 and IL-12, respectively. We confirmed the association of the IL-12A rs485497 polymorphism and pSS and found an increased serum protein level of IL-12p70 in pSS patients carrying the risk allele (p=0.016). Serum level of IL-12p70 was greater in patients than controls (p=0.0001), especially patients with more active disease (p=0.05); conversely IL-35 level was decreased in patients (p=0.0001) especially in patients with a more active disease (p=0.05). In blood cellular subsets, both IL-12p35 and EBI-3 mRNAs were detected only in B cells with a trend toward a lower level among pSS patients. Our findings emphasize the involvement of the IL-12/IL-35 balance in the pathogenesis of pSS. Serum IL-35 level was associated with low disease activity, in contrast to serum IL-12p70 level, which was rather associated with a more active disease. Copyright © 2017. Published by Elsevier Inc.

  5. Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

    PubMed

    Su, Bor Sheu; Yin, Hsien Sheng; Chiu, Hua Hsien; Hung, Li Hsiang; Huang, Ji Ping; Shien, Jui Hung; Lee, Long Huw

    2011-08-05

    Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.

  6. IL-12p40 gene-deficient BALB/c mice exhibit lower weight loss, reduced lung pathology and decreased sensitization to allergen in response to infection with pneumonia virus of mice.

    PubMed

    Shrivastava, Pratima; Sarkar, Indranil; Atanley, Ethel; Gomis, Susantha; van Drunen Littel-van den Hurk, Sylvia

    2016-10-01

    Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in infants and pneumonia virus of mice (PVM) causes similar disease. BALB/c mice are highly susceptible, while C57BL/6 mice are more resistant to PVM. IL-12 was significantly more up-regulated in response to PVM infection in BALB/c than in C57BL/6 mice. IL-12p40-deficient neonatal and adult BALB/c mice showed significantly less weight loss than wild-type mice after PVM challenge. The percentage of regulatory T cells, as well as IFN-β and IL-18 expression, was higher in the lungs of both neonatal and adult IL-12p40-/- mice. Adult IL-12p40-/- mice also showed enhanced TGF-β and IL-10 expression and reduced inflammatory responses. Furthermore, IL-12p40-/- mice showed decreased sensitization to inhaled cockroach antigen after PVM infection when compared to wild-type mice. In conclusion, these data suggest that a depressed regulatory capacity in BALB/c mice to PVM infection results in enhanced immunopathology and sensitization to allergen. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Delivery of IL-12 intranasally leads to reduced IL-12-mediated toxicity.

    PubMed

    Huber, Victor C; Arulanandam, Bernard P; Arnaboldi, Paul M; Elmore, Monica K; Sheehan, Christine E; Kallakury, Bhaskar V S; Metzger, Dennis W

    2003-06-01

    Interleukin-12 (IL-12) is a heterodimeric cytokine that enhances immune responses to bacterial, parasitic, and viral pathogens, and leads to tumor regression in animal models. For this reason, the use of IL-12 as a vaccine adjuvant and as a therapeutic agent for the treatment of cancer is being investigated. Unfortunately, the extreme toxicity of this molecule observed during clinical trials has limited its use. This toxicity correlates with increased IFN-gamma expression, decreased glucose levels, and altered histological responses in the spleen and duodenum. In this study, we show that intranasal (i.n.) delivery of IL-12 is a less toxic route of inoculation compared to the commonly employed subcutaneous route. When delivered i.n., IL-12 induces less systemic IFN-gamma production and fewer pathological tissue changes, yet is efficacious, as indicated by enhanced CD3(+) T cell activation and increased production of Th1-associated immunoglobulins (i.e., serum IgG2a). Thus, IL-12 can be delivered safely and effectively by the i.n. route, a finding which may allow IL-12 to fulfill its clinical potential.

  8. Epigenetic regulation of IL-12-dependent T cell proliferation

    PubMed Central

    Schaller, Matthew; Ito, Toshihiro; Allen, Ronald M.; Kroetz, Danielle; Kittan, Nicolai; Ptaschinski, Catherine; Cavassani, Karen; Carson, William F.; Godessart, Nuria; Grembecka, Jolanta; Cierpicki, Tomasz; Dou, Yali; Kunkel, Steven L.

    2015-01-01

    It is well established that the cytokine IL-12 and the transcription factor STAT4, an essential part of the IL-12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN-γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL-12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naïve T cell to a Th1 cell. Once MLL1 is up-regulated by IL-12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1+/− mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1+/−, T cells do not convert to a Th1 phenotype, as characterized by IFN-γ output. Furthermore, we observed a reduction in IFN-γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated. PMID:26059830

  9. Effect of IL12A and IL12B polymorphisms on the risk of Chlamydia trachomatis-induced tubal factor infertility and disease severity

    PubMed Central

    Öhman, H.; Bailey, R.; Natividad, A.; Ragoussis, J.; Johnson, L.-L.; Tiitinen, A.; Halttunen, M.; Paavonen, J.; Surcel, H.-M.

    2012-01-01

    BACKGROUND Interleukin-12 (IL-12) and related cytokines induce activation and differentiation of T cells. Our aim was to investigate the associations between genetic differences in IL-12-family cytokines and the pathogenesis of chlamydial disease. METHODS The final study population consisted of 100 women with Chlamydia trachomatis-induced tubal factor infertility (TFI) and 125 pregnant women as controls. Three single nucleotide polymorphisms (SNPs) of IL12A and seven SNPs of IL12B genes were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS We found that the IL12B SNP rs3212227 was associated with both susceptibility and severity of TFI. The minor allele C was rare and only one CC homozygote was found among the controls. AC heterozygotes were more common among TFI cases than among controls (P = 0.009) and were associated with increased risk of TFI [odds ratios (OR) = 2.44, 95% confidence intervals (CI) = 1.23–4.87]. Carrying the minor allele C was also associated with disease severity (P for trend = 0.008) and moderate (OR = 2.51, 95% CI = 1.06–5.95) and severe tubal damage (OR = 2.73, 95% CI = 1.15–6.52). CONCLUSIONS The results suggest that variation in the IL12B gene partly explains inter-individual differences in disease susceptibility and severity. PMID:22581790

  10. IL-12 is required for anti-OX40-mediated CD4 T cell survival.

    PubMed

    Ruby, Carl E; Montler, Ryan; Zheng, Rongxui; Shu, Suyu; Weinberg, Andrew D

    2008-02-15

    Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rbeta2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rbeta2 on OX40-stimulated CD4 T cells was tightly regulated and peaked approximately 4-6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rbeta2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.

  11. Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

    PubMed

    Marks, E; Naudin, C; Nolan, G; Goggins, B J; Burns, G; Mateer, S W; Latimore, J K; Minahan, K; Plank, M; Foster, P S; Callister, R; Veysey, M; Walker, M M; Talley, N J; Radford-Smith, G; Keely, S

    2017-01-25

    Intestinal inflammatory lesions are inherently hypoxic, due to increased metabolic demands created by cellular infiltration and proliferation, and reduced oxygen supply due to vascular damage. Hypoxia stabilizes the transcription factor hypoxia-inducible factor-1α (HIF) leading to a coordinated induction of endogenously protective pathways. We identified IL12B as a HIF-regulated gene and aimed to define how the HIF-IL-12p40 axis influenced intestinal inflammation. Intestinal lamina propria lymphocytes (LPL) were characterized in wild-type and IL-12p40(-/-) murine colitis treated with vehicle or HIF-stabilizing prolyl-hydroxylase inhibitors (PHDi). IL12B promoter analysis was performed to examine hypoxia-responsive elements. Immunoblot analysis of murine and human LPL supernatants was performed to characterize the HIF/IL-12p40 signaling axis. We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 and Th17 responses in murine colitis models. In the absence of IL-12p40, PHDi-treatment was ineffective. Analysis of the IL12B promoter identified canonical HIF-binding sites. HIF stabilization in LPLs resulted in production of IL-12p40 homodimer which was protective against colitis. The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This HIF-IL12p40 axis may represent an endogenously protective mechanism to limit the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.Mucosal Immunology advance online publication, 25 January 2017; doi:10.1038/mi.2016.135.

  12. Revisiting Human IL-12Rβ1 Deficiency

    PubMed Central

    de Beaucoudrey, Ludovic; Samarina, Arina; Bustamante, Jacinta; Cobat, Aurélie; Boisson-Dupuis, Stéphanie; Feinberg, Jacqueline; Al-Muhsen, Saleh; Jannière, Lucile; Rose, Yoann; Desurenaim, Maylis; Kong, Xiao-Fei; Filipe-Santos, Orchidée; Chapgier, Ariane; Picard, Capucine; Fischer, Alain; Dogu, Figen; Ikinciogullari, Aydan; Tanir, Gonul; Hajjar, Sami Al; Jumaah, Suliman Al; Frayha, Husn H.; AlSum, Zobida; Ajaji, Sulaiman Al; Alangari, Abdullah; Al-Ghonaium, Abdulaziz; Adimi, Parisa; Mansouri, Davood; Mustapha, Imen Ben; Yancoski, Judith; Garty, Ben Zion; Rodriguez-Gallego, Carlos; Caragol, Isabel; Kutukculer, Necil; Kumararatne, Dinanthaka S.; Patel, Smita; Doffinger, Rainer; Exley, Andrew; Jeppsson, Olle; Reichenbach, Janine; Nadal, David; Boyko, Yaryna; Pietrucha, Barbara; Anderson, Suzanne; Levin, Michael; Schandené, Liliane; Schepers, Kinda; Efira, André; Mascart, Françoise; Matsuoka, Masao; Sakai, Tatsunori; Siegrist, Claire-Anne; Frecerova, Klara; Blüetters-Sawatzki, Renate; Bernhöft, Jutta; Freihorst, Joachim; Baumann, Ulrich; Richter, Darko; Haerynck, Filomeen; De Baets, Frans; Novelli, Vas; Lammas, David; Vermylen, Christiane; Tuerlinckx, David; Nieuwhof, Chris; Pac, Malgorzata; Haas, Walther H.; Müller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Levy, Jacob; Raj, Revathi; Cohen, Aileen Cleary; Lewis, David B.; Holland, Steven; Yang, Kuender D.; Wang, Xiaochuan; Jiang, Xiaohong Wang, Liping; Yang, Xiqiang; Zhu, Chaomin; Xie, Yuanyuan; Lee, Pamela Pui Wah; Chan, Koon Wing; Chen, Tong-Xin; Castro, Gabriela; Ivelisse, Natera; Codoceo, Ana; King, Alejandra; Bezrodnik, Liliana; Giovani, Daniela Di; Gaillard, Maria Isabel; de Moraes-Vasconcelos, Dewton; Grumach, Anete Sevciovic; Duarte, Alberto Jose da Silva; Aldana, Ruth; Espinosa-Rosales, Francisco Javier; Bejaoui, Mohammed; Bousfiha, Ahmed Aziz; El Baghdadi, Jamila; Özbek, Namik; Aksu, Guzide; Keser, Melike; Somer, Ayper; Hatipoglu, Nevin; Aydogmus, Çigdem; Asilsoy, Suna; Camcioglu, Yildiz; Gülle, Saniye; Ozgur, Tuba T.; Ozen, Meteran; Oleastro, Matias; Bernasconi, Andrea; Mamishi, Setareh; Parvaneh, Nima; Rosenzweig, Sergio; Barbouche, Ridha; Pedraza, Sigifredo; Lau, Yu Lung; Ehlayel, Mohammad S.; Fieschi, Claire; Abel, Laurent; Sanal, Ozden; Casanova, Jean-Laurent

    2011-01-01

    Interleukin-12 receptor β1 (IL-12Rβ1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rβ1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought. PMID:21057261

  13. Natural killer cell depletion confounds the antitumor mechanism of endogenous IL-12 overexpression.

    PubMed

    Miller, George; Bleier, Joshua I; Antonescu, Cristina; Pillarisetty, Venu G; Shah, Alaap B; Lahrs, Svenja; DeMatteo, Ronald P

    2004-06-20

    IL-12 gene transfer to hepatocytes using a recombinant adenovirus vector (AdIL-12) has been shown to protect against primary and metastatic liver tumors in mice. However, the mechanism of protection has been elusive and studies using depleting monoclonal antibodies or transgenic mice have purported it to be independent of T and NK cells. We postulated that depletion of NK cells may distort the experimental model and misrepresent the antitumor mechanism by altering the magnitude and duration of transgene expression. We show in mice treated with AdIL-12 that NK depletion increased serum IL-12 levels by more than 250-fold and prolonged transgene expression by nearly 2 weeks compared to nondepleted mice. To determine the contribution of NK cells to tumor protection after AdIL-12 treatment, we analyzed NK cells from treated animals. Isolated NK cells were markedly activated in terms of their lytic activity and IFN-gamma secretion. Adoptive transfer of NK cells from mice that had been treated with AdIL-12 to naive mice was sufficient to confer protection against colorectal hepatic metastases. This protection was mediated in part by NK-cell production of IFN-gamma. Our findings indicate that NK-cell depletion distorts the model of systemic AdIL-12 administration by markedly altering transgene expression, which then may potentiate other antitumor mechanisms, and that endogenous IL-12 overexpression activates NK cells, rendering them sufficient to protect against liver metastases. These data have critical implications for investigating the immunologic mechanisms of experimental models that utilize gene transfer. Copyright 2004 Wiley-Liss, Inc.

  14. Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα

    PubMed Central

    Choi, I-K; Lee, J-S; Zhang, S-N; Park, J; Lee, K-M; Sonn, C H; Yun, C-O

    2011-01-01

    The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity. PMID:21451575

  15. Evaluation of γ-retroviral vectors that mediate the inducible expression of IL-12 for clinical application.

    PubMed

    Zhang, Ling; Feldman, Steven A; Zheng, Zhili; Chinnasamy, Nachimuthu; Xu, Hui; Nahvi, Azam V; Dudley, Mark E; Rosenberg, Steven A; Morgan, Richard A

    2012-06-01

    The clinical application of interleukin-12 (IL-12) has been hindered by the toxicity associated with its systemic administration. To potentially overcome this problem, we developed a promoter designed to direct IL-12 expression within the tumor environment using an inducible composite promoter containing binding motifs for the nuclear factor of activated T cells (NFAT) linked to a minimal IL-2 promoter. In this study, the NFAT promoter was coupled to a single-chain human IL-12 gene and inserted into 2 γ-retroviral self-inactivating vectors (SERS.NFAT.hIL12 and SERS.NFAT.hIL12.PA2) and 1 γ-retroviral vector (MSGV1.NFAT.hIL.12 PA2). Peripheral blood lymphocytes (PBLs) were double transduced with an antigen-specific T-cell receptor and the 3 NFAT.hIL12 vectors. Evaluation of inducible IL-12 expression, transduction efficiency, and vector production considerations led to the choice of the MSGV1.NFAT.hIL12.PA2 vector for clinical application. MSGV1.NFAT.hIL12.PA2 PG13 retroviral vector producer cell clones were screened by transduction of tumor antigen-specific PBLs. On the basis of expression studies in PBL, clone D3 was chosen to produce clinical-grade viral vector supernatant and was demonstrated to efficiently transduce young tumor-infiltrating lymphocytes (TIL). The vector-transduced young TIL with known tumor recognition demonstrated specific inducible IL-12 production after coculture with HLA-matched tumor targets and had augmented effector function as demonstrated by increased IFN-γ secretion. These results support the clinical application of adoptive transfer of young TIL engineered with the NFAT.hIL12 vector as a new approach for cancer immunotherapy.

  16. Primary Biliary Cirrhosis Associated with HLA, IL12A, and IL12RB2 Variants

    PubMed Central

    Hirschfield, Gideon M.; Liu, Xiangdong; Xu, Chun; Lu, Yue; Xie, Gang; Lu, Yan; Gu, Xiangjun; Walker, Erin J.; Jing, Kaiyan; Juran, Brian D.; Mason, Andrew L.; Myers, Robert P.; Peltekian, Kevork M.; Ghent, Cameron N.; Coltescu, Catalina; Atkinson, Elizabeth J.; Heathcote, E. Jenny; Lazaridis, Konstantinos N.; Amos, Christopher I.; Siminovitch, Katherine A.

    2010-01-01

    BACKGROUND Primary biliary cirrhosis is a chronic granulomatous cholangitis, characteristically associated with antimitochondrial antibodies. Twin and family aggregation data suggest that there is a significant genetic predisposition to primary biliary cirrhosis, but the susceptibility loci are unknown. METHODS To identify genetic loci conferring a risk for primary biliary cirrhosis, we carried out a genomewide association analysis in which DNA samples from 2072 Canadian and U.S. subjects (536 patients with primary biliary cirrhosis and 1536 controls) were genotyped for more than 300,000 single-nucleotide polymorphisms (SNPs). Sixteen of the SNPs most strongly associated with primary biliary cirrhosis were genotyped in two independent replication sets. We carried out fine-mapping studies across three loci associated with primary biliary cirrhosis. RESULTS We found significant associations between primary biliary cirrhosis and 13 loci across the HLA class II region; the HLA-DQB1 locus (encoding the major histocompatibility complex class II, DQ β chain 1) had the strongest association (P = 1.78×10−19; odds ratio for patients vs. controls, 1.75). Primary biliary cirrhosis was also significantly and reproducibly associated with two SNPs at the IL12A locus (encoding interleukin-12α), rs6441286 (P = 2.42×10−14; odds ratio, 1.54) and rs574808 (P = 1.88×10−13; odds ratio, 1.54), and one SNP at the IL12RB2 locus (encoding interleukin-12 receptor β2), rs3790567 (P = 2.76×10−11; odds ratio, 1.51). Fine-mapping analysis showed that a five-allele haplotype in the 3′ flank of IL12A was significantly associated with primary biliary cirrhosis (P = 1.15×10−34). We found a modest genomewide association (P<5.0×10−5) with the risk of disease for SNPs at the STAT4 locus (encoding signal transducer and activator of transcription 4) and the CTLA4 locus (encoding cytotoxic T-lymphocyte–associated protein 4) and 10 other loci. CONCLUSIONS Our data show significant

  17. New insights into IL-12-mediated tumor suppression

    PubMed Central

    Tugues, S; Burkhard, S H; Ohs, I; Vrohlings, M; Nussbaum, K; vom Berg, J; Kulig, P; Becher, B

    2015-01-01

    During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects. PMID:25190142

  18. New insights into IL-12-mediated tumor suppression.

    PubMed

    Tugues, S; Burkhard, S H; Ohs, I; Vrohlings, M; Nussbaum, K; Vom Berg, J; Kulig, P; Becher, B

    2015-02-01

    During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects.

  19. IL-1 beta induces dendritic cells to produce IL-12.

    PubMed

    Wesa, A K; Galy, A

    2001-08-01

    The cytokine IL-12, a product of dendritic cells (DC), plays a major role in cellular immunity, notably by inducing lymphocytes to produce IFN-gamma. Microbial products, T cell signals and cytokines induce the production of IL-12. Here, IL-1 beta is identified as a new IL-12-inducing agent, acting conjointly with CD40 ligand (CD40L) on human monocyte-derived DC in vitro. The effects of IL-1 beta were dose dependent, specifically blocked by neutralizing antibodies, and were observed both in immature and mature DC. Immature DC secreted more IL-12 than mature DC, but the effects of IL-1 beta were not due to a block of DC maturation as determined by analysis of DC surface markers. The mechanisms of action of IL-1 beta could be contrasted to that of other inducers of IL-12 such as IFN-gamma and lipopolysaccharide (LPS). Either IL-1 beta or IFN-gamma co-induced IL-12 with CD40L but conjointly, IL-1 beta, CD40L and IFN-gamma synergized, inducing very high levels of IL-12. The effects of IL-1 beta differed from those of LPS in that IL-1 beta, unlike LPS, could not induce IL-12 solely after IFN-gamma priming; and when combined with CD40L, IL-1 beta, unlike LPS, induced little IL-10. The mechanism of action of IL-1 beta involves IL-12 alpha mRNA up-regulation, and we show that the combination of CD40L and IL-1 beta induces high levels of IL-12 alpha and IL-12 beta mRNA in DC. Altogether, these results delineate a new mechanism linking adaptive and innate immune responses for the regulation of IL-12 production in DC and for the role of IL-1 beta in the development of cellular immunity.

  20. Systemic IL-12 Administration Alters Hepatic Dendritic Cell Stimulation Capabilities

    PubMed Central

    Chan, Tim; Back, Timothy C.; Subleski, Jeffrey J.; Weiss, Jonathan M.; Ortaldo, John R.; Wiltrout, Robert H.

    2012-01-01

    The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2–3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections. PMID:22428016

  1. IL-12 involvement in myogenic differentiation of C2C12 in vitro.

    PubMed

    Romanazzo, Sara; Forte, Giancarlo; Morishima, Keisuke; Taniguchi, Akiyoshi

    2015-03-01

    Recently, the extracellular microenvironment has been shown to be critical for the correct differentiation of stem cells to specific tissues. Many factors, including physical (e.g. biomaterial stiffness and topography) and biological (as growth factors, cytokines and chemokines) components, cooperate to create an ideal microenvironment for muscle stem cells, with many of these factors having been widely investigated. We previously demonstrated that the use of non-proliferating muscle-specific and unrelated cells as feeder layers for skeletal muscle progenitor cell differentiation resulted in significant differences in the ability to form myotubes, suggesting the importance of biological factors in myogenic differentiation. In this study, we investigated the biological factors involved in this process, analyzing the expression profile of 84 genes coding for cytokines and chemokines. We successfully identified a novel role for the cytokine IL-12 in the myogenic differentiation of C2C12 mouse skeletal muscle cells. Experiments involving the overexpression or silencing of the IL-12 gene in C2C12 showed that IL-12 enhanced the myogenic differentiation process. Moreover, when IL-12 was overexpressed in non-biologically related feeder cells, the new co-culture system was able to improve myogenic differentiation of C2C12 seeded on top. Although IL-12 is known to be a cytokine involved in inflammatory responses, it also appears to be involved in the myogenic differentiation process, acting as a positive regulator of this mechanism. This fact is expected to prove to be important for the development of functional biomaterials.

  2. Plasmid IL-12 electroporation in melanoma

    PubMed Central

    Cha, Edward; Daud, Adil

    2012-01-01

    Intratumoral gene electroporation uses electric charges to facilitate entry of plasmid DNA into cells in a reproducible and highly efficient manner, especially to accessible sites such as cutaneous and subcutaneous melanomas. Effective for locally treated disease, electroporation of plasmid DNA encoding interleukin-12 can also induce responses in untreated distant disease, suggesting that adaptive immune responses are being elicited that can target melanoma-associated antigens. In vivo electroporation with immunomodulatory cytokine DNA is a promising approach that can trigger systemic anti-tumor immune responses without the systemic toxicity associated with intravenous cytokine delivery and potentially offer complete long-term tumor regression. PMID:23151447

  3. Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

    PubMed Central

    Shan, Juanjuan; Shen, Junjie; Liu, Limei; Xu, Yanmin; Xia, Feng; Bie, Ping; Zhang, Xia; Cui, Youhong; Bian, Xiu-wu; Qian, Cheng

    2012-01-01

    Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells in vitro than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12. PMID:23028626

  4. Diverse Genome-wide Association Studies Associate the IL12/IL23 Pathway with Crohn Disease

    PubMed Central

    Wang, Kai; Zhang, Haitao; Kugathasan, Subra; Annese, Vito; Bradfield, Jonathan P.; Russell, Richard K.; Sleiman, Patrick M.A.; Imielinski, Marcin; Glessner, Joseph; Hou, Cuiping; Wilson, David C.; Walters, Thomas; Kim, Cecilia; Frackelton, Edward C.; Lionetti, Paolo; Barabino, Arrigo; Van Limbergen, Johan; Guthery, Stephen; Denson, Lee; Piccoli, David; Li, Mingyao; Dubinsky, Marla; Silverberg, Mark; Griffiths, Anne; Grant, Struan F.A.; Satsangi, Jack; Baldassano, Robert; Hakonarson, Hakon

    2009-01-01

    Previous genome-wide association (GWA) studies typically focus on single-locus analysis, which may not have the power to detect the majority of genuinely associated loci. Here, we applied pathway analysis using Affymetrix SNP genotype data from the Wellcome Trust Case Control Consortium (WTCCC) and uncovered significant association between Crohn Disease (CD) and the IL12/IL23 pathway, harboring 20 genes (p = 8 × 10−5). Interestingly, the pathway contains multiple genes (IL12B and JAK2) or homologs of genes (STAT3 and CCR6) that were recently identified as genuine susceptibility genes only through meta-analysis of several GWA studies. In addition, the pathway contains other susceptibility genes for CD, including IL18R1, JUN, IL12RB1, and TYK2, which do not reach genome-wide significance by single-marker association tests. The observed pathway-specific association signal was subsequently replicated in three additional GWA studies of European and African American ancestry generated on the Illumina HumanHap550 platform. Our study suggests that examination beyond individual SNP hits, by focusing on genetic networks and pathways, is important to unleashing the true power of GWA studies. PMID:19249008

  5. Polymorphisms of IL12RB2 May Affect the Natural History of Primary Biliary Cholangitis: A Single Centre Study

    PubMed Central

    Wasik, Urszula; Bogdanos, Dimitrios P.

    2017-01-01

    Background. Recent GWAS in primary biliary cholangitis (PBC) showed strong associations with SNPs located within interleukin-12 receptor (IL12R) beta-2 (IL12RB2) gene. Aims. We assessed whether genetic variation of IL12RB2 is associated with laboratory and clinical features of PBC. Methods. Genomic DNA was isolated from 306 patients with PBC and 258 age/gender-matched controls. PBC-specific anti-mitochondrial antibodies (AMA) were tested in all subjects by ELISA. Two SNPs, rs3790567 and rs6679356, of IL12RB2 were genotyped using the MGB-TaqMan SNP assay. Results. Despite comparable age at diagnosis of cirrhotic and noncirrhotic PBC patients, allele A of rs3790567 and allele C of rs6679356 were overrepresented in the former rather than the latter group (p = 0.0009 and p = 0.002, resp.). The risk of cirrhosis at presentation increased when allele A and allele C coexisted. AMA-M2 titres were significantly higher in AA homozygotes of rs3790567 compared to GG homozygotes (132 ± 54 versus 103 ± 62, p = 0.02) and in rs6679356 when C allele was present (p = 0.038). There were no other significant associations between IL12RB2 polymorphisms and laboratory or clinical features. Conclusion. In this first study analyzing phenotypic features of PBC carriers of the IL12RB2 polymorphisms, we found that carriers are more frequently cirrhotic at diagnosis and have significantly higher titres of AMA. PMID:28299343

  6. Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults.

    PubMed

    Phawong, Chintana; Ouma, Collins; Tangteerawatana, Piyatida; Thongshoob, Jarinee; Were, Tom; Mahakunkijcharoen, Yuvadee; Wattanasirichaigoon, Duangrurdee; Perkins, Douglas Jay; Khusmith, Srisin

    2010-06-01

    Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3' UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai-Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-gamma (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94-5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17-9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-gamma (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3' UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29-0.90; p = 0.020) and reduced circulating IFN-gamma (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3' UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-gamma levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-gamma levels.

  7. Macrophage IL-12p70 Signaling Prevents HSV-1–Induced CNS Autoimmunity Triggered by Autoaggressive CD4+ Tregs

    PubMed Central

    Mott, Kevin R.; Gate, David; Zandian, Mandana; Allen, Sariah J.; Rajasagi, Naveen Kumar; van Rooijen, Nico; Chen, Shuang; Arditi, Moshe; Rouse, Barry T.; Flavell, Richard A.; Town, Terrence; Ghiasi, Homayon

    2011-01-01

    Purpose. CD4+CD25+FoxP3+ naturally occurring regulatory T cells (Tregs) maintain self-tolerance and function to suppress overly exuberant immune responses. However, it is unclear whether innate immune cells modulate Treg function. Here the authors examined the role of innate immunity in lymphomyeloid homeostasis. Methods. The involvement of B cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and T cells in central nervous system (CNS) demyelination in different strains of mice infected ocularly with herpes simplex virus type 1 (HSV-1) was investigated. Results. The authors found that depletion of macrophages, but not DCs, B cells, NK cells, CD4+ T cells, or CD8+ T cells, induced CNS demyelination irrespective of virus or mouse strain. As with macrophage depletion, mice deficient in interleukin (IL)-12p35 or IL-12p40 showed CNS demyelination after HSV-1 infection, whereas demyelination was undetectable in HSV-1–infected, IL-23p19–deficient, or Epstein-Barr virus–induced gene 3-deficient mice. Demyelination could be rescued in macrophage-depleted mice after the injection of IL-12p70 DNA and in IL-12p35−/− or IL-12p40−/− mice after injection with IL-12p35 or IL-12p40 DNA or with recombinant viruses expressing IL-12p35 or IL-12p40. Using FoxP3-, CD4-, CD8-, or CD25-depletion and gene-deficient mouse approaches, the authors demonstrated that HSV-1–induced demyelination was blocked in the absence of CD4, CD25, or FoxP3 in macrophage-depleted mice. Flow cytometry showed an elevation of CD4+CD25+FoxP3+ T cells in the spleens of infected macrophage-depleted mice, and adoptive transfer of CD4+CD25+ T cells to infected macrophage-depleted severe combined immunodeficient mice induced CNS demyelination. Conclusions. The authors demonstrated that macrophage IL-12p70 signaling plays an important role in maintaining immune homeostasis in the CNS by preventing the development of autoaggressive CD4+ Tregs. PMID:21220560

  8. Congenital IL-12R1β receptor deficiency and thrombophilia in a girl homozygous for an IL12RB1 mutation and compound heterozygous for MTFHR mutations: A case report and literature review

    PubMed Central

    Kose, M.; Ceylan, O.; Patiroglu, T.; Bustamante, J.; Casanova, J. L.; Akyildiz, B. N.; Doganay, S.

    2014-01-01

    Interleukin-12 (IL-12) plays an important role in the production of interferon gamma from T cells and natural killer cells and is essential for protection against intra-macrophagic pathogens such as Mycobacterium and Salmonella. Here, we describe a 16-year-old girl with homozygous mutation in exon 12 of the IL12RB1 gene, which causes complete IL-12Rβ1 deficiency in association with heterozygous mutation (C677T and A1298C) in the methylenetetrahydrofolate reductase gene. She presented with disseminated Mycobacterium tuberculosis complex infection, retroperitoneal fungal abscess and also thrombosis in the superior mesenteric–portal vein junction. This is the first case report of a primary immunodeficiency associated with a genetically determined venous thrombosis. PMID:24678409

  9. Inherited IL-12p40 deficiency: genetic, immunologic, and clinical features of 49 patients from 30 kindreds.

    PubMed

    Prando, Carolina; Samarina, Arina; Bustamante, Jacinta; Boisson-Dupuis, Stéphanie; Cobat, Aurelie; Picard, Capucine; AlSum, Zobaida; Al-Jumaah, Suliman; Al-Hajjar, Sami; Frayha, Husn; Alangari, Abdullah; Al-Mousa, Hamoud; Mobaireek, Khalid F; Ben-Mustapha, Imen; Adimi, Parisa; Feinberg, Jacqueline; de Suremain, Maylis; Jannière, Lucile; Filipe-Santos, Orchidée; Mansouri, Nahal; Stephan, Jean-Louis; Nallusamy, Revathy; Kumararatne, Dinakantha S; Bloorsaz, Mohamad Reza; Ben-Ali, Meriem; Elloumi-Zghal, Houda; Chemli, Jalel; Bouguila, Jihene; Bejaoui, Mohamed; Alaki, Emadia; AlFawaz, Tariq S; Al Idrissi, Eman; ElGhazali, Gehad; Pollard, Andrew J; Murugasu, Belinda; Wah Lee, Bee; Halwani, Rabih; Al-Zahrani, Mohammed; Al Shehri, Mohammed A; Al-Zahrani, Mofareh; Bin-Hussain, Ibrahim; Mahdaviani, Seyed Alireza; Parvaneh, Nima; Abel, Laurent; Mansouri, Davood; Barbouche, Ridha; Al-Muhsen, Saleh; Casanova, Jean-Laurent

    2013-03-01

    Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is a rare genetic etiology of mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunologic, and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia, and Tunisia). There are only 9 different mutant alleles of the IL12B gene: 2 small insertions, 3 small deletions, 2 splice site mutations, and 1 large deletion, each causing a frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacille Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (n = 3), nocardiosis (n = 2), and klebsiellosis (n = 1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor β1 (IL-12Rβ1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients with MSMD

  10. Comparative antitumor effect among GM-CSF, IL-12 and GM-CSF+IL-12 genetically modified tumor cell vaccines.

    PubMed

    Miguel, A; Herrero, M J; Sendra, L; Botella, R; Algás, R; Sánchez, M; Aliño, S F

    2013-10-01

    Genetically modified cells have been shown to be one of the most effective cancer vaccine strategies. An evaluation is made of the efficacy of both preventive and therapeutic antitumor vaccines against murine melanoma, using C57BL/6 mice and irradiated B16 tumor cells expressing granulocyte and macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12) or both. Tumor was transplanted by the injection of wild-type B16 cells. Tumor growth and survival were measured to evaluate the efficacy of vaccination. Specific humoral response and immunoglobulin G (IgG) switch were evaluated measuring total IgG and IgG1 and IgG2a subtypes against tumor membrane proteins of B16 cells. In preventive vaccination, all treated groups showed delayed tumor growth. In addition, the group vaccinated to express only GM-CSF achieved 100% animal survival (P<0.005). Vaccination with GM-CSF+IL-12-producing B16 cells yielded lesser results (60% survival, P<0.005). Furthermore, all surviving animals remained disease-free after second tumor implantation 1 year later. The therapeutic vaccination strategies resulted in significantly delayed tumor growth, mainly using B16 cells producing GM-CSF+IL-12 cytokines, with 70% tumor growth inhibition (P<0.001)-although none of the animals reached overall survival. The results obtained suggest that the GM-CSF+IL-12 combination only increases the efficacy of therapeutic vaccines. No differences in classical regulatory T cells were found among the different groups.

  11. Morphine withdrawal inhibits IL-12 induction in a macrophage cell line through a mechanism that involves cAMP.

    PubMed

    Kelschenbach, Jennifer; Ninkovic, Jana; Wang, Jinghua; Krishnan, Anitha; Charboneau, Richard; Barke, Roderick A; Roy, Sabita

    2008-03-15

    There are very few studies that examine the effects that morphine withdrawal has on immune functioning, and of these even fewer describe the mechanisms by which withdrawal brings about these changes. Our previous work demonstrated that morphine withdrawal contributed to Th cell differentiation by biasing cells toward the Th2 lineage. A major finding from these studies was that IL-12 was decreased following withdrawal, and it was concluded that this decrease may be a mechanism by which morphine withdrawal is mediating Th2 polarization. Therefore, it was the aim of the current studies to develop an in vitro model to examine the process of morphine withdrawal and to understand the signaling mechanisms that withdrawal may use to effect IL-12 production through the use of this model. It was demonstrated and concluded that morphine withdrawal may be effecting IL-12 production by increasing cAMP levels, which activates protein kinase A. Protein kinase A activation then prevents the phosphorylation and subsequent degradation of IkappaB, which in turn prevents translocation of the NF-kappaB p65 subunit to the nucleus to transactivate the IL-12 p40 gene, ultimately resulting in decreased IL-12 production following LPS stimulation.

  12. Feline Leukemia Virus DNA Vaccine Efficacy Is Enhanced by Coadministration with Interleukin-12 (IL-12) and IL-18 Expression Vectors

    PubMed Central

    Hanlon, Linda; Argyle, David; Bain, Derek; Nicolson, Lesley; Dunham, Stephen; Golder, Matthew C.; McDonald, Michael; McGillivray, Christine; Jarrett, Oswald; Neil, James C.; Onions, David E.

    2001-01-01

    The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-γ). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-γ, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection. PMID:11507187

  13. IκBζ is essential for natural killer cell activation in response to IL-12 and IL-18

    PubMed Central

    Miyake, Tohru; Satoh, Takashi; Kato, Hiroki; Matsushita, Kazufumi; Kumagai, Yutaro; Vandenbon, Alexis; Tani, Tohru; Muta, Tatsushi; Akira, Shizuo; Takeuchi, Osamu

    2010-01-01

    IκBζ, encoded by Nfibiz, is a nuclear IκB-like protein harboring ankyrin repeats. IκBζ has been shown to regulate IL-6 production in macrophages and Th17 development in T cells. However, the role of IκBζ in natural killer (NK) cells has not be understood. In the present study, we found that the expression of IκBζ was rapidly induced in response to IL-18 in NK cells, but not in T cells. Analysis of Nfkbiz−/− mice revealed that IκBζ was essential for the production of IFN-γ production and cytotoxic activity in NK cells in response to IL-12 and/or IL-18 stimulation. IL-12/IL-18–mediated gene induction was profoundly impaired in Nfkbiz−/− NK cells. Whereas the phosphorylation of STAT4 was normally induced by IL-12 stimulation, STAT4 was not recruited to the Ifng gene regions in Nfkbiz−/− NK cells. Acetylation of histone 3 K9 on Ifng regions was also abrogated in Nfkbiz−/− NK cells. IκBζ was recruited on the proximal promoter region of the Ifng gene, and overexpression of IκBζ together with IL-12 activated the Ifng promoter. Furthermore, Nfkbiz−/− mice were highly susceptible to mouse MCMV infection. Taken together, these results demonstrate that IκBζ is essential for the activation of NK cells and antiviral host defense responses. PMID:20876105

  14. Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses.

    PubMed

    Sela, Uri; Park, Chae Gyu; Park, Andrew; Olds, Peter; Wang, Shu; Steinman, Ralph M; Fischetti, Vincent A

    2016-01-01

    Cytokines secreted from dendritic cells (DCs) play an important role in the regulation of T helper (Th) cell differentiation and activation into effector cells. Therefore, controlling cytokine secretion from DCs may potentially regulate Th differentiation/activation. DCs also induce de-novo generation of regulatory T cells (Treg) that modulate the immune response. In the current study we used the mixed leukocyte reaction (MLR) to investigate the effect of allospecific Treg on IL-12, TNFα and IL-6 secretion by DCs. Treg cells were found to markedly down-regulate IL-12 secretion from DCs following stimulation with TLR7/8 agonist. This down-regulation of IL-12 was neither due to a direct suppression of its production by the DCs nor a result of marked DC death. We found that IL-12 was rather actively consumed by Treg cells. IL-12 consumption was mediated by a subpopulation of IL-12Rβ2-expressing Treg cells and was dependent on MHC class-II expressed on dendritic cells. Furthermore, IL-12 consumption by Tregs increased their suppressive effect on T cell proliferation and Th1 activation. These results provide a new pathway of Th1 response regulation where IL-12 secreted by DCs is consumed by a sub-population of IL-12Rβ2-expressing Treg cells. Consumption of IL-12 by Tregs not only reduces the availability of IL-12 to Th effector cells but also enhances the Treg immunosuppressive effect. This DC-induced IL-12Rβ2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.

  15. Meta-Analysis on Associations of RGS1 and IL12A Polymorphisms with Celiac Disease Risk

    PubMed Central

    Guo, Cong-Cong; Wang, Man; Cao, Feng-Di; Huang, Wei-Huang; Xiao, Di; Ye, Xing-Guang; Ou, Mei-Ling; Zhang, Na; Zhang, Bao-Huan; Liu, Yang; Yang, Guang; Jing, Chun-Xia

    2016-01-01

    The pathogenesis of celiac disease (CD) has been related to polymorphisms in the regulator of G-protein signaling 1 (RGS1) and interleukin-12 A (IL12A) genes, but the existing findings are inconsistent. Our aim is to investigate the associations of two single-nucleotide polymorphisms (SNPs) (rs2816316 in RGS1 and rs17810546 in IL12A) with CD risk using meta-analysis. We searched PubMed and Web of Science on RGS1 rs2816316 and IL12A rs17810546 with CD risk. Odds ratio (OR) and 95% confidence interval (CI) of each SNP were estimated. All statistical analyses were performed on Stata 12.0. A total of seven studies were retrieved and analyzed. The available data indicated the minor allele C of rs2816316 was negatively associated with CD (C vs. A: OR = 0.77, 95% CI = 0.74–0.80), and a positive association was found for the minor allele G of rs17810546 (G vs. A: OR = 1.37, 95% CI = 1.31–1.43). The co-dominant model of genotype effect confirmed the significant associations between RGS1 rs2816316/IL12A rs17810546 and CD. No evidence of publication bias was observed. Our meta-analysis supports the associations of RGS1 and IL12A with CD and strongly calls for further studies to better understand the roles of RGS1 and IL12A in the pathogenesis of CD. PMID:27043536

  16. Role of IL-12 in Staphylococcus aureus-triggered arthritis and sepsis

    PubMed Central

    Hultgren, Olof H; Stenson, Martin; Tarkowski, Andrzej

    2001-01-01

    The present study demonstrates that endogenous production of IL-12 is crucial for survival in Staphylococcus aureus-induced arthritis in mice. Staphylococcal load is enhanced in several organs, because of lack of IL-12. This might be due to decreased production of IFN-γ in IL-12-deficient mice. Although IL-12-deficient mice were exposed to higher staphylococcal load, they demonstrated no increased severity of arthritis as compared with control animals. PMID:11178125

  17. Intratumoral immunotherapy of established solid tumors with chitosan/IL-12.

    PubMed

    Zaharoff, David A; Hance, Kenneth W; Rogers, Connie J; Schlom, Jeffrey; Greiner, John W

    2010-09-01

    IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicities after systemic administration. We hypothesized that intratumoral (i.t.) administration of IL-12 coformulated with the biodegradable polysaccharide chitosan could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Noninvasive imaging studies monitored local retention of IL-12, with and without chitosan coformulation, after i.t. injection. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established colorectal (MC32a) and pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i.t. injections of IL-12 alone eradicated ≤10% of established MC32a and Panc02 tumors, while i.t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4(+) or Gr-1(+) cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8(+) or NK cell depletion completely abrogated antitumor activity. I.t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as >80% of mice cured with i.t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity. Chitosan/IL-12 is a well-tolerated, effective immunotherapy with considerable potential for clinical translation.

  18. IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis

    PubMed Central

    Reeme, Allison E.; Miller, Halli E.; Robinson, Richard T.

    2015-01-01

    Summary IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CDllchi cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11chi lineages are absent. PMID:23491716

  19. The immunocytokine NHS-IL12 as a potential cancer therapeutic

    PubMed Central

    Kradjian, Giorgio; Guzman, Wilson; Bernhardt, Anna; Neuteboom, Berend; Lan, Yan; Sabzevari, Helen; Schlom, Jeffrey; Greiner, John W.

    2014-01-01

    Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy. PMID:24681847

  20. IL12Rβ1: The cytokine receptor that we used to know

    PubMed Central

    Robinson, Richard T.

    2014-01-01

    Human IL12RB1 encodes IL12Rβ1, a type I transmembrane receptor that is an essential component of the IL12- and IL23-signaling complex. IL12RB1 is well-established as being a promoter of delayed type hypersensitivity (DTH), the immunological reaction that limits tuberculosis. However, recent data demonstrate that in addition to promoting DTH, IL12RB1 also promotes autoimmunity. The contradictory roles of IL12RB1 in human health raises the question, what are the factors governing IL12RB1 function in a given individual, and how is inter-individual variability inIL12RB1 function introduced? Here we review recent data that demonstrate individual variability in IL12RB1 function is introduced at the epigenetic, genomic polymorphism, and mRNA splicing levels. Where and how these differences contribute to disease susceptibility and outcome are also reviewed. Collectively, recent data support a model whereinIL12RB1 sequence variability – whether introduced at the genomic or post-transcriptional level - contributes to disease, and that human IL12RB1 is not as simple agene as we once believed. PMID:25516297

  1. Expression of bioactive single-chain murine IL-12 in transgenic plants.

    PubMed

    Liu, Jianyun; Dolan, Maureen C; Reidy, Michael; Cramer, Carole L

    2008-06-01

    Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic. However, its clinical application is limited in part by lack of an effective bioproduction system for this complex heterodimeric glycoprotein. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. To test the potential of plants to effectively produce bioactive IL-12, we developed transgenic tobacco plant lines and derived root cultures yielding high levels of mouse IL-12 (MuIL-12). Functional IL-12 is a heterodimer consisting of two disulfide-linked subunits, p35 and p40. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of MuIL-12. Plant-derived single-chain MuIL-12 was characterized and purified for in vitro bioactivity assays. Our results demonstrated precise cleavage of the endogenous mouse p40 signal peptide in plants as well as addition of N-linked glycans. Plant-derived MuIL-12 triggered induction of interferon-gamma (IFN-gamma) secretion from mouse splenocytes and stimulated splenocyte proliferation with comparable activities to those observed for commercially available animal cell-derived MuIL-12. These studies indicate that plants produce fully functional MuIL-12 at levels compatible with commercial production and may serve as an effective bioproduction platform for bioactive IL-12s from other species for human or veterinary vaccine and therapeutic applications.

  2. IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis.

    PubMed

    Reeme, Allison E; Miller, Halli E; Robinson, Richard T

    2013-05-01

    IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CD11c(hi) cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11c(hi) lineages are absent.

  3. Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design

    PubMed Central

    Moafi, Mohammad; Rezvan, Hossein; Sherkat, Roya; Taleban, Roya; Asilian, Ali; Zarkesh Esfahani, Seyed Hamid; Nilforoushzadeh, Mohammad Ali; Jaffary, Fariba; Feizi, Awat

    2017-01-01

    Introduction Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. Methods and analysis This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rβ1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. Discussion It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. Ethics and dissemination This study has been approved by a local ethical committee. The final results will be

  4. Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design.

    PubMed

    Moafi, Mohammad; Rezvan, Hossein; Sherkat, Roya; Taleban, Roya; Asilian, Ali; Zarkesh Esfahani, Seyed Hamid; Nilforoushzadeh, Mohammad Ali; Jaffary, Fariba; Feizi, Awat

    2017-01-27

    Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rβ1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. This study has been approved by a local ethical committee. The final results will be disseminated through peer-reviewed journals and scientific conferences

  5. Innate Defects of the IL-12/IFN-γ Axis in Susceptibility to Infections by Mycobacteria and Salmonella

    PubMed Central

    Ramirez-Alejo, Noé

    2014-01-01

    Since 1996, several studies characterizing the association between primary immunodeficiencies and susceptibility to infections with environmental and non-pathogenic mycobacteria such as the Bacillus Calmette-Guérin (Mycobacterium bovis Bacillus of Calmette Guérin strain) as well as disseminated infections by Salmonella spp. have been conducted. These conditions, grouped in the so-called Mendelian susceptibility to mycobacterial diseases, include a primary immunodeficiency caused by mutations in 7 autosomal genes (IFNGR1, IFNGR2, IL12B, IL12BR1, STAT1, ISG15, and IRF8) and an X-linked gene (NEMO). This syndrome presents a high degree of allelic heterogeneity and variable penetrance. This review focuses on the analysis of the first reported cases of these diseases, as well as on the molecular findings involved in each of them. PMID:24359575

  6. IL-12Rβ2 has a protective role in relapsing-remitting experimental autoimmune encephalomyelitis

    PubMed Central

    Xie, Chong; Ciric, Bogoljub; Yu, Shuo; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2016-01-01

    IL-12Rβ2 participates in the receptors of IL-12 and IL-35, two cytokines that are involved in a variety of immune responses. In this study we evaluate the role of IL-12Rβ2 in relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that the IL-12Rβ2 deficient SJL/J EAE mice presented more severe symptoms and had more frequent, more severe relapses compared with wild type controls. IL-12Rβ2 deficient EAE mice also had more infiltrating mononuclear cells in the central nervous system, as well as higher splenic proliferative capacity and decreased IFN-γ production at the periphery. These findings suggest a protective role of IL-12Rβ2 in RR-EAE, an animal model of RR-MS, the most prevalent form of MS. PMID:26857496

  7. Serum amyloid A is an endogenous ligand that differentially induces IL-12 and IL-23.

    PubMed

    He, Rong; Shepard, Larry W; Chen, Jia; Pan, Zhixing K; Ye, Richard D

    2006-09-15

    The acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. However, their precise role in immunity and inflammation remains undefined. We report in this study a novel property of SAA in the differential induction of Th1-type immunomodulatory cytokines IL-12 and IL-23. In peripheral blood monocytes and the THP-1 monocytic cell line, SAA induces the expression of IL-12p40, a subunit shared by IL-12 and IL-23. SAA-stimulated expression of IL-12p40 was rapid (< or = 4 h), sustainable (> or = 20 h), potent (up to 3380 pg/ml/10(6) cells in 24 h), and insensitive to polymyxin B treatment. The SAA-stimulated IL-12p40 secretion required de novo protein synthesis and was accompanied by activation of the transcription factors NF-kappaB and C/EBP. Expression of IL-12p40 required activation of the p38 MAPK and PI3K. Interestingly, the SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. These results identify SAA as an endogenous ligand that potentially activates the IL-23/IL-17 pathway and present a novel mechanism for regulation of inflammation and immunity by an acute-phase protein.

  8. Preclinical validation: LV/IL-12 transduction of patient leukemia cells for immunotherapy of AML

    PubMed Central

    Huang, Ju; Liu, Yuanfeng; Au, Bryan C; Barber, Dwayne L; Arruda, Andrea; Schambach, Axel; Rothe, Michael; Minden, Mark D; Paige, Christopher J; Medin, Jeffrey A

    2016-01-01

    Interleukin-12 (IL-12) is a potent cytokine that may be harnessed to treat cancer. To date, nearly 100 IL-12-based clinical trials have been initiated worldwide. Yet systemic administration of IL-12 is toxic. Different strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Our previous studies employed lentivector-mediated expression of murine IL-12 in tumor cells and demonstrated effective protection in both mouse leukemia and solid tumor challenge models. In this study, we carried out preclinical validation studies using a novel lentivector to engineer expression of human IL-12 in acute myeloid leukemia blast cells isolated from 21 patients. Acute myeloid leukemia cells were transduced with a bicistronic lentivector that encodes the human IL-12 cDNA as a fusion, as well as a LNGFR (ΔLNGFR)/mutant thymidylate kinase cassette as a marking and cell-fate control element. A range of 20–70% functional transduction efficiencies was achieved. Transduced acute myeloid leukemia cells produced bioactive IL-12 protein and displayed dose-dependent sensitivity to the prodrug 3′-azido-3′-deoxythymidine. In vitro immortalization assays using transduced mouse hematopoietic stem cells demonstrated minimal genotoxic risk from our IL-12 vector. Scale-up transduction and cell processing was subsequently validated in a GMP facility to support our (now approved) Clinical Trial Application (CTA). PMID:27933304

  9. Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

    PubMed

    Aparicio-Siegmund, Samadhi; Moll, Jens M; Lokau, Juliane; Grusdat, Melanie; Schröder, Jutta; Plöhn, Svenja; Rose-John, Stefan; Grötzinger, Joachim; Lang, Philipp A; Scheller, Jürgen; Garbers, Christoph

    2014-01-01

    The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

  10. Modulation of IL-12 and IFNγ by probiotic supplementation promotes protection against Toxocara canis infection in mice.

    PubMed

    de Avila, L F D C; de Leon, P M M; de Moura, M Q; Berne, M E A; Scaini, C J; Leivas Leite, F P

    2016-05-01

    In this study, supplementation with the probiotic Saccharomyces boulardii promoted a reduction in intensity of infection by Toxocara canis and modulates cytokines mRNA expression in experimentally infected mice. IL-12 gene transcription had 40-fold increase in S. boulardii supplemented uninfected mice and sevenfold increase in supplemented infected mice comparing with not supplemented group. Regarding IFNγ, similar results were observed, since probiotic supplementation induced approximately 43-fold increase, but only in uninfected mice (P < 0·05). T. canis infection upregulated IL-10 expression while S. boulardii downregulated it and no change was observed for IL-4. Thus, based in these findings; we suggest that one possible mechanism responsible for S. boulardii protection effect against T. canis infection is by the modulation of cytokines expression, especially IL-12.

  11. Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members.

    PubMed

    Dixon, Karen O; van der Kooij, Sandra W; Vignali, Dario A A; van Kooten, Cees

    2015-06-01

    IL-35 is a cytokine of the IL-12 family, existing as a heterodimer of IL-12p35 and Ebi3. IL-35 has anti-inflammatory properties and is produced by regulatory T cells in humans and mice, where it is required for optimal suppression of immune responses. Distinct from other IL-12 cytokines, the expression of IL-35 has not been described in antigen-presenting cells. In view of the immune-regulatory properties of IL-35, we investigated the expression, regulation, and function of IL-12p35 and Ebi3 in human monocyte-derived dendritic cells and tolerogenic DCs (tolDCs). These tolDCs do not produce IL-12p70 or the homodimer IL-12p40. We demonstrate that tolDCs completely lack transcriptional expression of IL-12p40. However, tolDCs maintain mRNA expression of IL-12p35 and Ebi3. Using intracellular flow cytometry and Western blot analysis, we show that tolDCs produce Ebi3 and IL-12p35, and both can be enhanced upon stimulation with IFN-γ, LPS, or CD40L. tolDCs supernatants have the capacity to suppress T-cell activation. Using IL12A silencing, we demonstrate that IL-12p35 is required for tolDCs to reach their full suppressive potential. Taken together, our results indicate that tolDCs produce IL-35, providing an additional novel mechanism by which tolDCs elicit their tolerogenic potential. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Contribution of IL-12/IL-35 Common Subunit p35 to Maintaining the Testicular Immune Privilege

    PubMed Central

    Terayama, Hayato; Yoshimoto, Takayuki; Hirai, Shuichi; Naito, Munekazu; Qu, Ning; Hatayama, Naoyuki; Hayashi, Shogo; Mitobe, Kana; Furusawa, Jun-ichi; Mizoguchi, Izuru; Kezuka, Takeshi; Goto, Hiroshi; Suyama, Kaori; Moriyama, Hiroshi; Sakabe, Kou; Itoh, Masahiro

    2014-01-01

    The testis is an organ with immune privilege. The comprehensive blood–testis barrier formed by Sertoli cells protects autoimmunogenic spermatozoa and spermatids from attack by the body’s immune system. The interleukin (IL)-6/IL-12 family cytokines IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Epstein-Barr virus−induced gene 3 [EBI3]), and IL-35 (p35/EBI3) play critical roles in the regulation of various immune responses, but their roles in testicular immune privilege are not well understood. In the present study, we investigated whether these cytokines are expressed in the testes and whether they function in the testicular immune privilege by using mice deficient in their subunits. Expression of EBI3 was markedly increased at both mRNA and protein levels in the testes of 10- or 12-week-old wild-type mice as compared with levels in 2-week-old mice, whereas the mRNA expression of p40 was markedly decreased and that of p35 was conserved between these two groups. Lack of EBI3, p35, and IL-12 receptor β2 caused enhanced infiltration of lymphocytes into the testicular interstitium, with increased interferon-γ expression in the testes and autoantibody production against mainly acrosomal regions of spermatids. Spermatogenic disturbance was more frequently observed in the seminiferous tubules, especially when surrounded by infiltrating lymphocytes, of these deficient mice than in those of wild-type mice. In particular, p35-deficient mice showed the most severe spermatogenic disturbance. Immunohistochemical analyses revealed that endothelial cells and peritubular cells in the interstitium were highly positive for p35 at both ages, and CD163+ resident macrophages positive for p35 and EBI3, possibly producing IL-35, were also detected in the interstitium of 12-week-old mice but not those of 2-week-old mice. These results suggest that p35 helps in maintaining the testicular immune privilege, in part in an IL-35-dependent manner. PMID:24760014

  13. Interleukin-12 (IL-12), but not IL-23, deficiency ameliorates viral encephalitis without affecting viral control.

    PubMed

    Kapil, Parul; Atkinson, Roscoe; Ramakrishna, Chandran; Cua, Daniel J; Bergmann, Cornelia C; Stohlman, Stephen A

    2009-06-01

    The relative contributions of interleukin-12 (IL-12) and IL-23 to viral pathogenesis have not been extensively studied. IL-12p40 mRNA rapidly increases after neurotropic coronavirus infection. Infection of mice defective in both IL-12 and IL-23 (p40(-/-)), in IL-12 alone (p35(-/-)), and in IL-23 alone (p19(-/-)) revealed that the symptoms of coronavirus-induced encephalitis are regulated by IL-12. IL-17-producing cells never exceeded background levels, supporting a redundant role of IL-23 in pathogenesis. Viral control, tropism, and demyelination were all similar in p35(-/-), p19(-/-), and wild-type mice. Reduced morbidity in infected IL-12 deficient mice was also not associated with altered recruitment or composition of inflammatory cells. However, gamma interferon (IFN-gamma) levels and virus-specific IFN-gamma-secreting CD4 and CD8 T cells were all reduced in the central nervous systems (CNS) of infected p35(-/-) mice. Transcription of the proinflammatory cytokines IL-1beta and IL-6, but not tumor necrosis factor, were initially reduced in infected p35(-/-) mice but increased to wild-type levels during peak inflammation. Furthermore, although transforming growth factor beta mRNA was not affected, IL-10 was increased in the CNS in the absence of IL-12. These data suggest that IL-12 does not contribute to antiviral function within the CNS but enhances morbidity associated with viral encephalitis by increasing the ratio of IFN-gamma to protective IL-10.

  14. Additive effects of exogenous IL-12 supplementation and antibiotic treatment in infection prophylaxis.

    PubMed

    Boyce, Brandon M; Lindsey, Brock A; Clovis, Nina B; Smith, E Suzanne; Hobbs, Gerald R; Hubbard, David F; Emery, Sanford E; Barnett, John B; Li, Bingyun

    2012-02-01

    The increasing clinical incidence and host risk of open fracture-associated infections, as well as the reduced effectiveness of conventional antibiotics to treat such infections, have driven the development of new therapies for the prophylaxis of open fracture-associated infections. We investigated percutaneous supplementation of a natural cytokine (i.e., interleukin 12p70 or IL-12) at an open fracture site to reduce open fracture-associated infections. We also determined the efficacy of the combination therapy of IL-12 and conventional antibiotic therapy in the prophylaxis of open fracture-associated infections. An open femur fracture infection model was produced by direct inoculation of a clinical isolate of Staphylococcus aureus after creating a femur fracture using rats. The animals were assigned to one of four groups: no drug administration, percutaneous supplementation of IL-12, intraperitoneal administration of the antibiotic ampicillin, or percutaneous IL-12 in combination with intraperitoneal ampicillin. Animals were euthanized at postoperative days 6, 10, 14, and 21. Percutaneous IL-12 led to a reduction in infection at postoperative days 6 and 10. For the first time, exogenous IL-12 was found to have additive effects in the prevention of infection when combined with conventional treatment (i.e., antibiotic therapy). Combination therapy of ampicillin and IL-12 substantially reduced the infection rate at postoperative day 6 and also decreased the time needed for complete inhibition of infection. Therefore, exogenous IL-12, providing a mechanism of protection independent of antibiotic resistance, complements the routine use of antibiotics.

  15. Inhibitory effects of IL-12 on experimental tooth movement and root resorption in mice.

    PubMed

    Yoshimatsu, Masako; Kitaura, Hideki; Fujimura, Yuji; Kohara, Haruka; Morita, Yukiko; Eguchi, Toshiko; Yoshida, Noriaki

    2012-01-01

    Interleukin (IL)-12 is an important cytokine for innate and adaptive immunity. We previously reported that IL-12 inhibits tumour necrosis factor (TNF)-α-mediated osteoclast formation by inducing apoptosis. We also reported that TNF-α plays an important role in mechanical loading-induced osteoclast formation and bone resorption during orthodontic tooth movement. In this study, we investigated the effects of IL-12 on mechanical tooth movement in mice. A Ni-Ti closed coil spring was inserted between the upper incisors and the upper left first molar in mice. IL-12 was injected locally adjacent to the first molar every other day during the experimental period, at doses varying from 0 to 1.5μg/day. After 12 days, the animals were killed and their jaws were processed for histological evaluation using tartrate-resistant acid phosphatase (TRAP) and TdT-mediated dUTP-biotin nick end-labelling (TUNEL) staining, and measurements of the root resorption area. In the IL-12-treated mice, tooth movement and root resorption appeared to be reduced. In TUNEL-stained sections, many apoptotic cells were recognized on the pressure side in the IL-12-treated mice. Our findings suggest that IL-12 inhibits not only mechanical tooth movement, but also root resorption during orthodontic tooth movement. These findings may arise through apoptosis induced by IL-12. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. IL-12 receptor 1β deficiency with features of autoimmunity and photosensitivity.

    PubMed

    Ling, Galina; Ling, Eduard; Broides, Arnon; Poran Feldman, Hagit; Levy, Jacov; Garty, Ben-Zion; Nahum, Amit

    2016-01-01

    Primary immunodeficiences are often accompanied by autoimmune phenomena. IL-12 receptor deficiency is a well characterized primary immunodeficiency that leads to propensity to intracellular infections mainly with mycobacteria and Salmonella. We report on two patients with IL-12 receptor β1 deficiency that presented with autoimmune manifestations and photosensitivity dermatitis and describe possible pathogenetic mechanisms leading to development of clinically significant autoimmune phenomena.

  17. IFN-gamma-independent effects of IL-12 during intestinal nematode infection.

    PubMed

    Helmby, Helena; Grencis, Richard K

    2003-10-01

    Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with a pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. When exogenous rIL-12 was administered to T. spiralis-infected NIH mice, this resulted in significant suppression of intestinal mast cell responses, delayed worm expulsion, increased muscle larvae burdens, and a transient, but significant decrease in early Th2 cytokine secretion. rIL-12 treatment also altered chemokine expression in the jejunal mucosa. The effects of exogenous IL-12 administration were largely independent of IFN-gamma as shown by rIL-12 treatment of IFN-gamma knockout mice. Hence, IL-12 may play a significant biological role as a direct negative regulator of intestinal Th2 responses and may act to promote the survival of intestinal parasites in vivo also in the absence of IFN-gamma.

  18. IκBζ augments IL-12- and IL-18-mediated IFN-γ production in human NK cells.

    PubMed

    Kannan, Yashaswini; Yu, Jianhua; Raices, Raquel M; Seshadri, Sudarshan; Wei, Min; Caligiuri, Michael A; Wewers, Mark D

    2011-03-10

    Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18-induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56(+) NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56(-) lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56(+) cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes.

  19. IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression.

    PubMed

    Gil-Fariña, Irene; Di Scala, Marianna; Vanrell, Lucia; Olagüe, Cristina; Vales, Africa; High, Katherine A; Prieto, Jesus; Mingozzi, Federico; Gonzalez-Aseguinolaza, Gloria

    2013-01-01

    Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.

  20. Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

    PubMed Central

    Schilbach, Karin; Alkhaled, Mohammed; Welker, Christian; Eckert, Franziska; Blank, Gregor; Ziegler, Hendrik; Sterk, Marco; Müller, Friederike; Sonntag, Katja; Wieder, Thomas; Braumüller, Heidi; Schmitt, Julia; Eyrich, Matthias; Schleicher, Sabine; Seitz, Christian; Erbacher, Annika; Pichler, Bernd J; Müller, Hartmut; Tighe, Robert; Lim, Annick; Gillies, Stephen D; Strittmatter, Wolfgang; Röcken, Martin; Handgretinger, Rupert

    2015-01-01

    Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo. PMID:26140238

  1. A Novel Immunoregulatory Function for IL-23: Inhibition of IL-12 Dependent IFN-γ Production

    PubMed Central

    Sieve, Amy N.; Meeks, Karen D.; Lee, Suheung; Berg, Rance E.

    2011-01-01

    Summary Most studies investigating the function of IL-23 have concluded that it promotes IL-17 secreting T cells. While some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 antagonizes IL-12 induced secretion of IFN-γ. When splenocytes or purified populations of T cells are cultured with IL-23, IFN-γ secretion in response to IL-12 is dramatically reduced. The impact of IL-23 is most prominent in CD8 T cells, but is also observed in NK and CD4 T cells. Mechanistically, the IL-23 receptor is not required for this phenomenon, and IL-23 inhibits signaling through the IL-12 receptor by reducing IL-12 induced signal transducer and activator of transcription 4 (STAT4) phosphorylation. IL-23 is also able to reduce IFN-γ secretion by antagonizing endogenously produced IL-12 from Listeria monocytogenes (LM) infected macrophages. In vivo, LM infection induces higher serum IFN-γ levels and a greater percentage of IFN-γ+CD8+ T cells in IL-23p19 deficient mice as compared to wild-type mice. This increase in IFN-γ production coincides with increased LM clearance at days 2–3 post-infection. Our data suggest that IL-23 may be a key factor in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ. PMID:20458705

  2. IL-12Rβ2 is critical for survival of primary Francisella tularensis LVS infection

    PubMed Central

    Melillo, Amanda A.; Foreman, Oded; Elkins, Karen L.

    2013-01-01

    Using a panel of vaccines that provided different degrees of protection, we previously identified the IL-12 receptor subunit β2 as a mediator, whose relative expression correlated with strength of protection against secondary lethal challenge of vaccinated mice with an intracellular bacterium, the LVS of Francisella tularensis. The present study therefore tested the hypothesis that IL-12Rβ2 is an important mediator in resistance to LVS by directly examining its role during infections. IL-12Rβ2 KO mice were highly susceptible to LVS primary infection, administered i.d. or i.n. The LD50 of LVS infection of KO mice were 2 logs lower than those of WT mice, regardless of route. Five days after infection with LVS, bacterial organ burdens were significantly higher in IL-12Rβ2 KO mice. IL-12Rβ2 KO mice infected with lethal doses of LVS had more severe liver pathology, including significant increases in the liver enzymes ALT and AST. Despite decreased levels of IFN-γ, LVS-vaccinated IL-12Rβ2 KO mice survived large lethal LVS secondary challenge. Consistent with in vivo protection, in vitro intramacrophage LVS growth was well-controlled in cocultures containing WT or IL-12Rβ2 KO LVS-immune splenocytes. Thus, survival of secondary LVS challenge was not strictly dependent on IL-12Rβ2. However, IL-12Rβ2 is important in parenteral and mucosal host resistance to primary LVS infection and in the ability of WT mice to clear LVS infection and serves to restrict liver damage. PMID:23440500

  3. The 3'UTR 1188A/C polymorphism of IL-12p40 is not associated with susceptibility for developing plaque psoriasis in Mestizo population from western Mexico.

    PubMed

    Sandoval-Talamantes, Ana Karen; Brito-Luna, Myrian Johanna; Fafutis-Morris, Mary; Villanueva-Quintero, Delfina Guadalupe; Graciano-Machuca, Omar; Ramírez-Dueñas, María Guadalupe; Alvarado-Navarro, Anabell

    2015-02-01

    Psoriasis is a chronic autoimmune inflammatory disease that affects the skin and the joints. Psoriasis is characterized by the keratinocyte proliferation, which is induced by cytokines Th1 and Th17. Patients with plaque psoriasis present a chronic inflammatory response with high levels of interleukin (IL)-12 and IL-23. Various single-nucleotide polymorphisms (SNP) have been identified in the IL12B gene, such as SNP 3' UTR 1188 A/C (SNP rs3212227), which has been associated with susceptibility to developing plaque psoriasis and with the production of IL-12 and IL-23 in individuals of different ethnic groups. In this study, we determined whether there is an association of SNP rs3212227 with the susceptibility of developing plaque psoriasis and with serum levels of IL-12 and IL-23 in Mestizo population in western Mexico. We included 112 patients with psoriasis and 112 clinical healthy individuals in the study. The frequencies of genotypes A/A, A/C, and C/C in patients with plaque psoriasis were 41, 53, and 6%, respectively, while in the control group, these were 37, 53, and 10%, respectively, without finding statistically significant differences between both groups (p>0.05). Although IL-12 and IL-23 serum levels were higher in patients than in controls, we found no significant differences. The group of patients with genotype CC presented the highest levels of IL-23 (p<0.05). These data suggest that the SNP rs3212227 phenotype is not associated with the risk of developing plaque psoriasis or with IL-12 and IL-23 levels in Mestizo population in western Mexico. Copyright © 2014 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  4. Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

    PubMed Central

    la Sala, Andrea; He, Jianping; Laricchia-Robbio, Leopoldo; Gorini, Stefania; Iwasaki, Akiko; Braun, Michael; Yap, George S.; Sher, Alan; Ozato, Keiko

    2009-01-01

    Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8. PMID:19487420

  5. Inherited disorders of the IL-12-IFN-gamma axis in patients with disseminated BCG infection.

    PubMed

    Mansouri, Davood; Adimi, Parisa; Mirsaeidi, Mehdi; Mansouri, Nahal; Khalilzadeh, Soheila; Masjedi, Mohammad R; Adimi, Parvaneh; Tabarsi, Payam; Naderi, Mohammad; Filipe-Santos, Orchidée; Vogt, Guillaume; de Beaucoudrey, Ludovic; Bustamante, Jacinta; Chapgier, Ariane; Feinberg, Jacqueline; Velayati, Ali A; Casanova, Jean-Laurent

    2005-12-01

    Disseminated BCG infection is a rare complication of vaccination that occurs in patients with impaired immunity. In recent years, a series of inherited disorders of the IL-12-IFN-gamma axis have been described that predispose affected individuals to disseminated disease caused by BCG, environmental Mycobacteria, and non-typhoidal Salmonella. The routine immunological work-up of these patients is normal and the diagnosis requires specific investigation of the IL-12-IFN-gamma circuit. We report here the first two such patients originating from and living in Iran. The first child is two years old and suffers from complete IFN-gamma receptor 2 deficiency and disseminated BCG infection. He is currently in clinical remission thanks to prolonged multiple antibiotic therapy. The other, a 28-year-old adult, suffers from IL-12p40 deficiency and presented with disseminated BCG infection followed by recurrent episodes of systemic salmonellosis. He is now doing well. A third patient of Iranian descent, living in North America, was reported elsewhere to suffer from IL-12Rbeta1 deficiency. These three patients thus indicate that various inherited defects of the IL-12-IFN-gamma circuit can be found in Iranian people. In conclusion we recommend to consider the disorders of the IL-12-IFN-gamma circuit in all patients with severe BCG infection, disseminated environmental mycobacterial disease, or systemic non-typhoidal salmonellosis, regardless of their ethnic origin and country of residence.

  6. IL-12 receptor β1 deficiency alters in vivo T follicular helper cell response in humans

    PubMed Central

    Bustamante, Jacinta; Bourdery, Laure; Bentebibel, Salah Eddine; Boisson-Dupuis, Stephanie; Hamlin, Fran; Tran, Mau V.; Blankenship, Derek; Pascual, Virginia; Savino, Daniel A.; Banchereau, Jacques; Casanova, Jean-Laurent

    2013-01-01

    Antibody responses represent a key immune protection mechanism. T follicular helper (Tfh) cells are the major CD4+ T-cell subset that provides help to B cells to generate an antibody response. Tfh cells together with B cells form germinal centers (GCs), the site where high-affinity B cells are selected and differentiate into either memory B cells or long-lived plasma cells. We show here that interleukin-12 receptor β1 (IL-12Rβ1)–mediated signaling is important for in vivo Tfh response in humans. Although not prone to B cell-deficient–associated infections, subjects lacking functional IL-12Rβ1, a receptor for IL-12 and IL-23, displayed substantially less circulating memory Tfh and memory B cells than control subjects. GC formation in lymph nodes was also impaired in IL-12Rβ1–deficient subjects. Consistently, the avidity of tetanus toxoid–specific serum antibodies was substantially lower in these subjects than in age-matched controls. Tfh cells in tonsils from control individuals displayed the active form of signal transducer and activator of transcription 4 (STAT4), demonstrating that IL-12 is also acting on Tfh cells in GCs. Thus, our study shows that the IL-12–STAT4 axis is associated with the development and the functions of Tfh cells in vivo in humans. PMID:23476048

  7. Influence of TYK2 in systemic sclerosis susceptibility: a new locus in the IL-12 pathway.

    PubMed

    López-Isac, Elena; Campillo-Davo, Diana; Bossini-Castillo, Lara; Guerra, Sandra G; Assassi, Shervin; Simeón, Carmen Pilar; Carreira, Patricia; Ortego-Centeno, Norberto; García de la Peña, Paloma; Beretta, Lorenzo; Santaniello, Alessandro; Bellocchi, Chiara; Lunardi, Claudio; Moroncini, Gianluca; Gabrielli, Armando; Riemekasten, Gabriela; Witte, Torsten; Hunzelmann, Nicolas; Kreuter, Alexander; Distler, Jörg Hw; Voskuyl, Alexandre E; de Vries-Bouwstra, Jeska; Herrick, Ariane; Worthington, Jane; Denton, Christopher P; Fonseca, Carmen; Radstake, Timothy Rdj; Mayes, Maureen D; Martín, Javier

    2016-08-01

    TYK2 is a common genetic risk factor for several autoimmune diseases. This gene encodes a protein kinase involved in interleukin 12 (IL-12) pathway, which is a well-known player in the pathogenesis of systemic sclerosis (SSc). Therefore, we aimed to assess the possible role of this locus in SSc. This study comprised a total of 7103 patients with SSc and 12 220 healthy controls of European ancestry from Spain, USA, Germany, the Netherlands, Italy and the UK. Four TYK2 single-nucleotide polymorphisms (V362F (rs2304256), P1104A (rs34536443), I684S (rs12720356) and A928V (rs35018800)) were selected for follow-up based on the results of an Immunochip screening phase of the locus. Association and dependence analyses were performed by the means of logistic regression and conditional logistic regression. Meta-analyses were performed using the inverse variance method. Genome-wide significance level was reached for TYK2 V362F common variant in our pooled analysis (p=3.08×10(-13), OR=0.83), while the association of P1104A, A928V and I684S rare and low-frequency missense variants remained significant with nominal signals (p=2.28×10(-3), OR=0.80; p=1.27×10(-3), OR=0.59; p=2.63×10(-5), OR=0.83, respectively). Interestingly, dependence and allelic combination analyses showed that the strong association observed for V362F with SSc, corresponded to a synthetic association dependent on the effect of the three previously mentioned TYK2 missense variants. We report for the first time the association of TYK2 with SSc and reinforce the relevance of the IL-12 pathway in SSc pathophysiology. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  8. IL-12 Enhances the Antitumor Actions of Trastuzumab via NK Cell IFN-γ Production

    PubMed Central

    Jaime-Ramirez, Alena Cristina; Mundy-Bosse, Bethany L.; Kondadasula, SriVidya; Jones, Natalie B.; Roda, Julie M.; Mani, Aruna; Parihar, Robin; Karpa, Volodymyr; Papenfuss, Tracey L.; LaPerle, Krista M.; Biller, Elizabeth; Lehman, Amy; Chaudhury, Abhik Ray; Jarjoura, David; Burry, Richard W.; Carson, William E.

    2013-01-01

    The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26HER2/neu) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ–deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4+ or CD8+ T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26HER2/neu tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs. PMID:21321106

  9. IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production.

    PubMed

    Jaime-Ramirez, Alena Cristina; Mundy-Bosse, Bethany L; Kondadasula, SriVidya; Jones, Natalie B; Roda, Julie M; Mani, Aruna; Parihar, Robin; Karpa, Volodymyr; Papenfuss, Tracey L; LaPerle, Krista M; Biller, Elizabeth; Lehman, Amy; Chaudhury, Abhik Ray; Jarjoura, David; Burry, Richard W; Carson, William E

    2011-03-15

    The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

  10. IL-12 Is an Effective Adjuvant to Recombinant Vaccinia Virus-Based Tumor Vaccines

    PubMed Central

    Rao, Jay B.; Chamberlain, Ronald S.; Bronte, Vincenzo; Carroll, Miles W.; Irvine, Kari R.; Moss, Bernard; Rosenberg, Steven A.; Restifo, Nicholas P.

    2007-01-01

    A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, β-galactosidase (β-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding β-gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag the costimulatory molecule B7-1, designated B7-1β-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor-bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines. PMID:8617961

  11. IL-12 and IL-18 levels in serum and gingival tissue in aggressive and chronic periodontitis.

    PubMed

    Sánchez-Hernández, P E; Zamora-Perez, A L; Fuentes-Lerma, M; Robles-Gómez, C; Mariaud-Schmidt, R P; Guerrero-Velázquez, C

    2011-07-01

    The aim of this study was to compare the levels of interleukin-12 (IL-12) and IL-18 in gingival tissue and serum between patients with chronic (n = 18) or aggressive periodontitis (n = 12) and healthy subjects (HS) (n = 9). Gingival tissue biopsies and serum were obtained from all study subjects. The tissue was homogenized and cytokines IL-12 and IL-18 were quantified by enzyme-linked immunosorbent assay. Interleukin-12 levels in gingival tissue were significantly higher in aggressive periodontitis patients than in HS; serum IL-12 was significantly elevated in aggressive periodontitis relative to both chronic periodontitis (CP) and HS. IL-18 levels in gingival tissue showed no significant differences between the groups. Patients with CP showed significantly elevated levels of serum IL-18 compared with HS; however, the aggressive periodontitis group showed no significant differences with either the CP group or the HS. Our results showed higher levels of IL-12 in gingival tissue and serum of patients with aggressive periodontitis, and IL-18 was elevated in the serum of CP patients. The patterns of IL-12 and IL-18 are different in chronic and aggressive periodontitis; this finding suggests distinctive mechanisms of immunopathogenesis between these forms of periodontitis. © 2011 John Wiley & Sons A/S.

  12. Lethal tuberculosis in a previously healthy adult with IL-12 receptor deficiency.

    PubMed

    Tabarsi, Payam; Marjani, Majid; Mansouri, Nahal; Farnia, Parisa; Boisson-Dupuis, Stephanie; Bustamante, Jacinta; Abel, Laurent; Adimi, Parisa; Casanova, Jean-Laurent; Mansouri, Davood

    2011-08-01

    A 33-year-old man was admitted in hospital due to fever, generalized lymphadenopathy, and hepatosplenomegaly. He had a history of anti-tuberculosis treatment in the previous 3 years. Despite normal chest radiograph, a sputum sample was smear-positive for acid-fast bacilli, and polymerase chain reaction was positive for Mycobacterium tuberculosis complex. Drug susceptibility test revealed resistance to isoniazid and rifampin. Evaluation of the patient's immune system revealed IL-12Rβ1 deficiency. The patient died of disseminated tuberculosis (TB), despite appropriate antibiotic treatment. This is the first IL-12 receptor-deficient patient presenting with disseminated TB in adulthood, without any previous relevant medical history. This diagnosis should be considered in selected adult patients with unexplained, overwhelming TB. IL-12Rβ1 deficiency is a genetic etiology of severe TB in adults and should be considered in adult patients with disseminated TB.

  13. Transient Expression of Transgenic IL-12 in Mouse Liver Triggers Unremitting Inflammation Mimicking Human Autoimmune Hepatitis.

    PubMed

    Gil-Farina, Irene; Di Scala, Marianna; Salido, Eduardo; López-Franco, Esperanza; Rodríguez-García, Estefania; Blasi, Mercedes; Merino, Juana; Aldabe, Rafael; Prieto, Jesús; Gonzalez-Aseguinolaza, Gloria

    2016-09-15

    The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encoding murine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, anti-nuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-γ remained elevated during the entire study period. IFN-γ was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies.

  14. MICA/IL-12: A novel bifunctional protein for killer cell activation.

    PubMed

    Tietje, Ashlee; Yang, Xi; Yu, Xianzhong; Wei, Yanzhang

    2017-03-01

    Natural killer (NK) cells have the potential to be effective killers of tumor cells. They are governed by inhibitory and activating receptors such as NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have ways to reduce the expression of these ligands, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, such as IL-12. A previous study in our laboratory showed that a fusion protein of the extracellular domain of mouse UL-16 binding protein-like transcript 1 (MULT1E) and mouse interleukin 12 (IL-12) can effectively activate mouse NK cells by in vitro assays and in vivo in animal tumor models. The aim of the present study was to expand the concept of developing a novel bifunctional fusion protein for enhanced NK cell activation to human killer cells. The proposed protein combines the extracellular domain of a human NKG2D ligand, MHC class I polypeptide-related sequence A (MICA) and IL-12. It is hypothesized that when expressed by tumor cells, the protein will activate human NK and other killer cells using the NKG2D receptor, and deliver IL-12 to the NK cells where it can interact with the IL-12R and enhance cytotoxicity. The fusion protein, when expressed by engineered tumor cells, indeed activated NK92 cells as measured by an increase in interferon-γ (IFN-γ) production and an increase in cytotoxicity of tumor cells. The fusion protein was also able to increase the proliferation of human peripheral blood mononuclear cells (PBMCs) and augment their production of IFN-γ. This study along with the data from the previous mouse studies suggest that the MICA/IL-12 bifunctional fusion protein represents an effective activator of killer cells for cancer treatment.

  15. Induction of IL-12 from human monocytes after stimulation with Androctonus crassicauda scorpion venom.

    PubMed

    Saadi, Samahir; Assarehzadegan, Mohammad-Ali; Pipelzadeh, Mohammad Hassan; Hadaddezfuli, Reza

    2015-11-01

    The objective of this study was to evaluate the capacity of venom from Androctonus crassicauda to induce expression/production of interleukin (IL)-12 by isolated human monocytes. For this purpose, isolated human monocytes were exposed to different concentrations of the venom (0.16-20 μg/ml) for varying periods (6, 12, and 24 h). Apart from measures of venom cytotoxicity (i.e., lactase dehydrogenase activity [LDH] release), measures of IL-12 p40 mRNA (by Real-time PCR) of IL-12 release (by ELISA) were performed. The results showed that the venom produced significant concentration- and duration of incubation-dependent cytotoxicity. Expression of IL-12 p40 mRNA was significantly increased at all exposure timepoints relative to that in unexposed cells, but was maximal after 6 h of exposure. At that timepoint, the effect from a dose of 2.5 μg venom/ml provided the maximal increase among all doses tested. At the level of the protein itself, IL-12 production remained almost consistently elevated (vs. unexposed control values) across all exposure timepoints, with the greatest formation again occurring after 6 h of incubation at a dose of 2.5 μg venom/ml. The findings from this study demonstrated that venom from the A. crassicauda scorpion contained active constituents that could induce a sustained activation of human monocytes that was manifested, in part, as promotion of the expression/production of IL-12. Copyright © 2015. Published by Elsevier Ltd.

  16. In vivo Antitumor Effect of an HPV-specific Promoter driving IL-12 Expression in an HPV 16-positive Murine Model of Cervical Cancer

    PubMed Central

    Bermúdez-Morales, Victor Hugo; Fierros-Zarate, Geny; García-Meléndrez, Celina; Alcocer-Gonzalez, Juan Manuel; Morales-Ortega, Ausencio; Peralta-Zaragoza, Oscar; Torres-Poveda, Kirvis; Burguete-García, Ana Isabel; Hernández-Márquez, Eva; Madrid-Marina, Vicente

    2016-01-01

    Human papillomavirus (HPV) is a DNA virus that infects epithelial cells and has been implicated in the development of cervical cancer. Few therapeutic strategies have been designed for the treatment of cervical intraepithelial neoplasia, a precursor of cervical cancer. In these early stages, the HPV E2 protein is the most important viral factor involved in viral gene expression and plays crucial roles during the vegetative viral cycle in epithelial cells. Papillomavirus E2 binds specifically to palindromic ACCN6GGT sequences, referred to as the E2 binding sites (E2BS), which are concentrated within the viral long control region, and which are responsible for regulation of the HPV protein's expression. Here, we consider E2BS as a candidate sequence to induce the expression of antiviral therapeutic genes selectively in HPV-infected cells expressing the E2 protein. This study focuses on the use of an HPV-specific promoter comprised of four E2BS to drive the expression of IL-12, leading to an antitumor effect in an HPV-positive murine tumor model. The therapeutic strategy was implemented via viral gene therapy using adenoviral vectors with recombinant E2 and IL-12 genes and E2BS-IL-12. We demonstrate that the HPV-specific promoter E2BS is functional in vitro and in vivo through transactivation of HPV E2 transcription factor. PMID:27877210

  17. Evaluation of local MCP-1 and IL-12 nanocoatings for infection prevention in open fractures.

    PubMed

    Li, Bingyun; Jiang, Bingbing; Dietz, Matthew J; Smith, E Suzanne; Clovis, Nina B; Rao, K Murali Krishna

    2010-01-01

    The increasing incidence of bacterial infection and the appearance of Staphylococcus aureus (S. aureus) strains that are resistant to commonly used antibiotics has made it important to develop non-antibiotic approaches for infection prevention. The aim of this study was to develop local monocyte chemoattractant protein-1 (MCP-1) and interleukin-12 p70 (IL-12 p70) therapies to prevent S. aureus infection by enhancing the recruitment and activation of macrophages, which are believed to play an important role in infection prevention as the first line of defense against invading pathogens. Nanocoating systems for MCP-1 and IL-12 p70 deliveries were prepared, and their release characteristics desirable for infection prevention in open fractures were explored. Local MCP-1 therapy reduced S. aureus infection and influenced white blood cell populations, and local IL-12 p70 treatment had a more profound effect on preventing S. aureus infection. No synergistic relationship in decreasing S. aureus infection was observed when MCP-1 and IL-12 p70 treatments were combined. This reported new approach may reduce antibiotic use and antibiotic resistance.

  18. Clinical and genetic features of IL12Rb1 deficiency: Single center experience of 18 patients.

    PubMed

    Tan, Çağman; Çağdaş-Ayvaz, Deniz; Metin, Ayşe; Keskin, Özlem; Tezcan, İlhan; Sanal, Özden

    2016-01-01

    Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by infections with weakly virulent mycobacteria (BCG and environmental mycobacteria), M. tuberculosis, Salmonella, candida and some other intracellular microorganisms. Nine different genetic defects have been defined to cause MSMD and IL-12Rβ1 deficiency is the most common form. We present here the clinical and genetic features of 18 patients with IL12Rβ1 deficiency diagnosed by surface expression of IL-12Rβ1 and Sanger's sequencing. Seventeen patients showed classical presentation (infections with BCG, salmonella and candida) while one patient experienced recurrent leishmaniasis. In all patients the percentage of activated lymphocytes with surface expression of IL12Rβ1 was < 1% indicating that it is an effective method for the screening of these patients. Three recurrent mutations were responsible for 85% of our families. Prognosis was good in patients, in whom specific antimicrobial therapy was given before dissemination occurs, as well as prophylactic antimicrobial treatment when needed and IFN-γ therapy for severe infectious episodes.

  19. Evaluation of local MCP-1 and IL-12 nanocoatings for infection prevention in open fractures#

    PubMed Central

    Li, Bingyun; Jiang, Bingbing; Dietz, Matthew J.; Smith, E. Suzanne; Clovis, Nina B.; Krishna Rao, K. Murali

    2013-01-01

    The increasing incidence of bacterial infection and the appearance of Staphylococcus aureus (S. aureus) strains that are resistant to commonly used antibiotics has made it important to develop non-antibiotic approaches for infection prevention. The aim of this study was to develop local monocyte chemoattractant protein-1 (MCP-1) and interleukin-12 p70 (IL-12 p70) therapies to prevent S. aureus infection by enhancing the recruitment and activation of macrophages, which are believed to play an important role in infection prevention as the first line of defense against invading pathogens. Nanocoating systems for MCP-1 and IL-12 p70 deliveries were prepared and their release characteristics desirable for infection prevention in open fractures were explored. Local MCP-1 therapy reduced S. aureus infection and influenced white blood cell populations, and local IL-12 p70 treatment had a more profound effect on preventing S. aureus infection. No synergistic relationship in decreasing S. aureus infection was observed when MCP-1 and IL-12 p70 treatments were combined. This reported new approach may reduce antibiotic use and antibiotic resistance. PMID:19588527

  20. Interleukins IL-6, IL-8, IL-10, IL-12 and periimplant disease. An update.

    PubMed

    Candel-Martí, Maria-Eugenia; Flichy-Fernández, Antonio-Juan; Alegre-Domingo, Teresa; Ata-Ali, Javier; Peñarrocha-Diago, Maria A

    2011-07-01

    A study is made of the usefulness of cytokines (such as interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-12 (IL-12)) as markers of periimplant disease (mucositis and periimplantitis). An increase in the levels of these cytokines in dental implant crevicular fluid may give rise to a lack of osteointegration, bone loss or implant failure. To review the literature relating IL-6, IL-8, IL-10 and IL-12 levels to dental implant surgery and periimplantitis. A PubMed literature search was made of articles in English and Spanish, using the key words "cytokine and dental implants", cytokine and periimplantitis", "IL-6, IL-8, IL-10, IL-12 and dental implants", "IL-6, IL-8, IL-10, IL-12 and periimplantitis". Fourteen articles were found and classified into two groups relating interleukin levels to: a) periimplant disease; and b) their influence upon dental implant osteointegration without periimplant disease. An increase in interleukin levels is observed in patients with periimplant disease, though there is controversy over the effect of interleukins in crevicular fluid and periimplantitis in relation to implant failure or the development of periimplant disease.

  1. Expanding Diversity in Molecular Structures and Functions of the IL-6/IL-12 Heterodimeric Cytokine Family

    PubMed Central

    Hasegawa, Hideaki; Mizoguchi, Izuru; Chiba, Yukino; Ohashi, Mio; Xu, Mingli; Yoshimoto, Takayuki

    2016-01-01

    The interleukin (IL)-6/IL-12 family cytokines have pleiotropic functions and play critical roles in multiple immune responses. This cytokine family has very unique characteristics in that they comprise two distinct subunits forming a heterodimer and each cytokine and receptor subunit shares with each other. The members of this cytokine family are increasing; currently, there are more than six cytokines, including the tentatively named cytokines IL-Y (p28/p40), IL-12 (p35/p40), IL-23 (p19/p40), IL-27 [p28/Epstein–Barr virus-induced protein 3 (EBI3)], IL-35 (p35/EBI3), and IL-39 (p19/EBI3). This family of cytokines covers a very broad range of immune responses, including pro-inflammatory responses, such as helper T (Th)1, Th2, and Th17, to anti-inflammatory responses, such as regulatory T (Treg) cells and IL-10-producing Treg cells. IL-12 is the first member of this family, and IL-12, IL-23, and IL-27 are mainly produced by activated antigen-presenting cells, such as dendritic cells and macrophages. IL-12 plays a critical role in the promotion of Th1 immune responses by inducing interferon-γ production to combat pathogens and malignant tumors. IL-23 induces IL-17 production and is necessary to maintain pathogenic Th17 cells that cause inflammatory and autoimmune diseases. IL-27 was initially reported to play a critical role in promotion of Th1 differentiation; however, subsequent studies revealed that IL-27 has broader stimulatory and inhibitory roles by inducing IL-10-producing Treg cells. IL-35 is produced by forkhead box P3+ Treg cells and activated B cells and has immunosuppressive functions to maintain immune tolerance. The most recently identified cytokine, IL-39, is produced by activated B cells and has pro-inflammatory functions. The cytokine tentatively named IL-Y seems to have anti-inflammatory functions by inhibiting Th1 and Th17 differentiation. In addition, individual cytokine subunits were also shown to have self-standing activities. Thus

  2. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 ± 1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  3. In vivo stimulation of IL-12 secretion by subcutaneous low-dose IL-2 in metastatic cancer patients.

    PubMed Central

    Lissoni, P.; Fumagalli, L.; Rovelli, F.; Brivio, F.; Di Felice, G.; Majorca, F.

    1998-01-01

    Despite the well-demonstrated involvement of both interleukin 2 (IL-2) and interleukin 12 (IL-12) in the activation of host anti-cancer response, the knowledge of IL-2-IL-12 interactions has still to be better investigated. This study was performed to evaluate the effects of subcutaneous (s.c.) low-dose IL-2 on IL-12 secretion in metastatic cancer patients. The study included 19 evaluable metastatic renal cell cancer patients, who received s.c. low-dose IL-2 (6 MIU day(-1) for 6 days per week for 4 weeks) as a first-line immunotherapy of their metastatic disease. Serum levels of IL-12 were measured using an enzyme immunoassay on venous blood samples collected before the immunotherapy and at 1-week intervals. The clinical response consisted of partial response (PR) in four and stable disease (SD) in eight patients, whereas the other seven patients progressed. Mean serum levels of IL-12 observed in the overall patients significantly increased in response to IL-2 injection. Moreover, by evaluating IL-12 variations in relation to the clinical response, a marked significant increase in IL-12 mean values occurred in patients with response or SD, whereas the progressing patients showed a significant decline in IL-12 levels during IL-2 administration. Finally, IL-12 mean pretreatment values observed in patients who progressed were significantly higher than those seen in non-progressing patients. This study shows that low-dose IL-2 immunotherapy of cancer may stimulate the in vivo release of IL-12, and it would suggest that IL-2-induced IL-12 enhancement is associated with a favourable prognosis. PMID:9667674

  4. In vivo stimulation of IL-12 secretion by subcutaneous low-dose IL-2 in metastatic cancer patients.

    PubMed

    Lissoni, P; Fumagalli, L; Rovelli, F; Brivio, F; Di Felice, G; Majorca, F

    1998-06-01

    Despite the well-demonstrated involvement of both interleukin 2 (IL-2) and interleukin 12 (IL-12) in the activation of host anti-cancer response, the knowledge of IL-2-IL-12 interactions has still to be better investigated. This study was performed to evaluate the effects of subcutaneous (s.c.) low-dose IL-2 on IL-12 secretion in metastatic cancer patients. The study included 19 evaluable metastatic renal cell cancer patients, who received s.c. low-dose IL-2 (6 MIU day(-1) for 6 days per week for 4 weeks) as a first-line immunotherapy of their metastatic disease. Serum levels of IL-12 were measured using an enzyme immunoassay on venous blood samples collected before the immunotherapy and at 1-week intervals. The clinical response consisted of partial response (PR) in four and stable disease (SD) in eight patients, whereas the other seven patients progressed. Mean serum levels of IL-12 observed in the overall patients significantly increased in response to IL-2 injection. Moreover, by evaluating IL-12 variations in relation to the clinical response, a marked significant increase in IL-12 mean values occurred in patients with response or SD, whereas the progressing patients showed a significant decline in IL-12 levels during IL-2 administration. Finally, IL-12 mean pretreatment values observed in patients who progressed were significantly higher than those seen in non-progressing patients. This study shows that low-dose IL-2 immunotherapy of cancer may stimulate the in vivo release of IL-12, and it would suggest that IL-2-induced IL-12 enhancement is associated with a favourable prognosis.

  5. Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model.

    PubMed

    Galvan, Daniel L; O'Neil, Richard T; Foster, Aaron E; Huye, Leslie; Bear, Adham; Rooney, Cliona M; Wilson, Matthew H

    2015-01-01

    Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

  6. Interleukin 12 (IL-12) Family Cytokines: Role in Immune Pathogenesis and Treatment of CNS Autoimmune Disease

    PubMed Central

    Sun, Lin; He, Chang; Nair, Lekha; Yeung, Justine; Egwuagu, Charles E.

    2015-01-01

    Cytokines play crucial roles in coordinating the activities of innate and adaptive immune systems. In response to pathogen recognition, innate immune cells secrete cytokines that inform the adaptive immune system about the nature of the pathogen and instruct naïve T cells to differentiate into the appropriate T cell subtypes required to clear the infection. These include Interleukins, Interferons and other immune-regulatory cytokines that exhibit remarkable functional redundancy and pleiotropic effects. The focus of this review, however, is on the enigmatic Interleukin 12 (IL-12) family of cytokines. This family of cytokines plays crucial roles in shaping immune responses during antigen presentation and influence cell-fate decisions of differentiating naïve T cells. They also play essential roles in regulating functions of a variety of effector cells, making IL-12 family cytokines important therapeutic targets or agents in a number of inflammatory diseases, such as the CNS autoimmune diseases, uveitis and multiple sclerosis. PMID:25796985

  7. Clinical Significance of Serum IL-12 Level in Patients with Early Breast Carcinoma and Its Correlation with Other Tumor Markers

    PubMed Central

    Youssef, Samar Samir; Mohammad, Manal Moussa; Ezz-El-Arab, Lobna R.

    2015-01-01

    AIM: To investigate the diagnostic significance of Interleukin 12 (IL-12) in breast cancer (BC) and its correlation with other tumor markers including cancer antigen 15-3 (CA 15-3), carcinoembryonic antigen (CEA), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MMP9/TIMP1 ratio. METHODS: Serum levels of IL-12, tumor markers, and hormone receptors were measured in 92 BC and 56 benign lesion patients versus 40 healthy subjects. Clinical stage, tumor size, lymph node metastasis, grade, and histological type were recorded. RESULTS: BC patients have lower IL-12, but higher CA 15.3 and CEA than control group. High levels of serum IL-12 were associated with lymph node positivity and progesterone receptor negativity. IL-12 was significant lower in invasive ductal carcinoma (IDC) compared to non IDC histological type. IL-12 was higher in patients with higher stage and grade but the difference was not statistically significant. IL-12 correlates negatively with MMP9/TIMP1 ratio. CONCLUSION: IL-12 is less specific than CEA for screening early BC, but its correlation with tumor aggressiveness and progression markers may have a prognostic value. PMID:27275301

  8. Isolation of IL-12p70-competent human monocyte-derived dendritic cells.

    PubMed

    Søndergaard, Jonas N; Brix, Susanne

    2012-12-14

    Diverse methodologies ranging from experimental immunological studies to immunotherapy involve the application of human monocyte-derived dendritic cells (moDCs). Considerable donor-dependent variations in the moDC production of IL-12p70 affect the outcome of these methodologies. It has been shown that moDCs generated under standard conditions develop into two subsets based on CD1a-expression with the CD1a+ moDCs being the main IL-12p70 producers. This has however not been generally accepted, which we show here because the subset described as CD1a-negative does express CD1a, but at a lower level than the other subset. We further characterize the phenotype of these two subsets, showing that the CD1a-hi subset has a greater immunogenic phenotype, making this subset more suitable for immunotherapy. The two subsets have previously been separated by cell sorting, but as this technique is not available to many laboratories and has incompatibility with clinical settings, a more widely useable technique is warranted. Therefore we tested if magnetic-activated cell sorting is useful for the purpose, and show that it is possible to isolate IL-12p70-competent CD1a-hi moDCs to a <92% purity, irrespective of the starting purity.

  9. Effect of IL-12 on canine dendritic cell maturation following differentiation induced by granulocyte-macrophage CSF and IL-4.

    PubMed

    Sugiura, Kikuya; Wijewardana, Viskam; Fujimoto, Mariko; Akazawa, Takashi; Yahata, Mana; Mito, Kai; Hatoya, Shingo; Inoue, Norimitsu; Inaba, Toshio

    2010-10-15

    IL-12 is a cytokine produced by dendritic cells (DC) and activates cytotoxic T cells and NK cells against tumors. We investigated the effect of IL-12 on DC. When peripheral blood monocytes were incubated with canine granulocyte-macrophage (GM)-CSF and IL-4, expression of MHC class II, CD1a, CD80 and CD86 was significantly elevated, but the cell morphology was that of immature DC. By adding canine IL-12 to the DC-inducing culture, expression of CD80 and CD1a was significantly enhanced, and the cell morphology shifted to that of mature DC. T cell stimulation by cultured cells was also significantly enhanced by the presence of IL-12. These results show that IL-12 significantly promotes the maturation and activation of canine DC following the induction of differentiation by GM-CSF and IL-4. (c) 2010 Elsevier B.V. All rights reserved.

  10. In vivo suppression of plasma IL-12 levels by acute and chronic stress paradigms: potential mediating mechanisms and sex differences.

    PubMed

    Shaashua, L; Sominsky, L; Levi, B; Sorski, L; Reznick, M; Page, G G; Ben-Eliyahu, S

    2012-08-01

    Interleukin-12 (IL-12) is a major pro-inflammatory cytokine, which promotes cell-mediated immunity and T(H)1 differentiation. In vitro studies indicated suppression of IL-12 production by several stress-related factors, but no effects of behavioral stress were shown on plasma IL-12 levels. Therefore, in the current study we (i) examined the in vivo effects of various behavioral and pharmacological stress paradigms on baseline plasma IL-12 levels; (ii) compared these in vivo findings to those obtained following in vitro stimulation of leukocytes from the same rats; and (iii) assessed potential sexual dimorphism in these outcomes. The findings indicated that plasma IL-12 levels were significantly reduced by social confrontation, wet-cage exposure, surgery, and the administration of corticosterone, epinephrine, or prostaglandin-E(2). Notably, most in vivo impacts on plasma levels were not evident when assessed in vitro. The IL-12-reducing effects of wet-cage exposure, and of corticosterone and epinephrine administration, were significantly greater in males than in females, although females exhibited greater total corticosterone levels following stress. The duration of acute stressors predicted the degree of IL-12 reduction, but more prolonged stressors did not. Furthermore, seven days of alternating behavioral stressors reduced plasma IL-12 levels more than 14 days. These findings suggest animals' behavioral habituation to stress conditions, or a specific immune mechanism restricting the duration of IL-12 reduction. Overall, our findings indicate a generic and robust stress-induced reduction in plasma IL-12 levels, and suggest epinephrine, corticosterone, and prostaglandin-E(2), as potential mediators that should be scrutinized in vivo in the context of natural physiological stress responses. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. In vivo suppression of plasma IL-12 levels by acute and chronic stress paradigms: potential mediating mechanisms and sex differences

    PubMed Central

    Shaashua, L.; Sominsky, L.; Levi, B.; Sorski, L.; Reznick, M.; Page, G.; Ben-Eliyahu, S.

    2012-01-01

    Interleukin-12 (IL-12) is a major pro-inflammatory cytokine, which promotes cell-mediated immunity and TH1 differentiation. In vitro studies indicated suppression of IL-12 production by several stress-related factors, but no effects of behavioral stress were shown on plasma IL-12 levels. Therefore, in the current study we (i) examined the in vivo effects of various behavioral and pharmacological stress paradigms on baseline plasma IL-12 levels; (ii) compared these in vivo findings to those obtained following in vitro stimulation of leukocytes from the same rats; and (iii) assessed potential sexual dimorphism in these outcomes. The findings indicated that plasma IL-12 levels were significantly reduced by social confrontation, wet-cage exposure, surgery, and the administration of corticosterone, epinephrine, or prostaglandin-E2. Notably, most in vivo impacts on plasma levels were not evident when assessed in vitro. The IL-12-reducing effects of wet-cage exposure, and of corticosterone and epinephrine administration, were significantly greater in males than in females, although females exhibited greater total corticosterone levels following stress. The duration of acute stressors predicted the degree of IL-12 reduction, but more prolonged stressors did not. Furthermore, seven days of alternating behavioral stressors reduced plasma IL-12 levels more than fourteen days. These findings suggest animals’ behavioral habituation to stress conditions, or a specific immune mechanism restricting the duration of IL-12 reduction. Overall, our findings indicate a generic and robust stress-induced reduction in plasma IL-12 levels, and suggest epinephrine, corticosterone, and prostaglandin-E2, as potential mediators that should be scrutinized in vivo in the context of natural physiological stress responses. PMID:22659252

  12. IL-12 and type I interferon prolong the division of activated CD8 T cells by maintaining high-affinity IL-2 signaling in vivo

    PubMed Central

    Starbeck-Miller, Gabriel R.; Xue, Hai-Hui

    2014-01-01

    TCR ligation and co-stimulation induce cellular division; however, optimal accumulation of effector CD8 T cells requires direct inflammatory signaling by signal 3 cytokines, such as IL-12 or type I IFNs. Although in vitro studies suggest that IL-12/type I IFN may enhance T cell survival or early proliferation, the mechanisms underlying optimal accumulation of CD8 T cells in vivo are unknown. In particular, it is unclear if disparate signal 3 cytokines optimize effector CD8 T cell accumulation by the same mechanism and how these inflammatory cytokines, which are transiently produced early after infection, affect T cell accumulation many days later at the peak of the immune response. Here, we show that transient exposure of CD8 T cells to IL-12 or type I IFN does not promote survival or confer an early proliferative advantage in vivo, but rather sustains surface expression of CD25, the high-affinity IL-2 receptor. This prolongs division of CD8 T cells in response to basal IL-2, through activation of the PI3K pathway and expression of FoxM1, a positive regulator of cell cycle progression genes. Thus, signal 3 cytokines use a common pathway to optimize effector CD8 T cell accumulation through a temporally orchestrated sequence of cytokine signals that sustain division rather than survival. PMID:24367005

  13. IκBζ augments IL-12– and IL-18–mediated IFN-γ production in human NK cells

    PubMed Central

    Kannan, Yashaswini; Yu, Jianhua; Raices, Raquel M.; Seshadri, Sudarshan; Wei, Min; Caligiuri, Michael A.

    2011-01-01

    Interferon-γ (IFN-γ) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of κB-ζ (IκBζ), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-γ production in KG-1 cells, we tested IκBζ's role in the classic lymphocyte pathway of IL-12/IL-18–induced IFN-γ. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of IκBζ and released IFN-γ. CD56+ NK cells were shown to be the IκBζ-producing lymphocyte subpopulation, which also released abundant IFN-γ in response to IL-12/IL-18. Importantly, IκBζ was undetectable in CD56− lymphocytes where IFN-γ release was 10-fold lower. In addition, small interfering RNA knockdown of IκBζ suppressed IFN-γ expression in CD56+ cells. The association of IκBζ with the IFN-γ promoter was documented by chromatin immunoprecipitation. IFN-γ promoter activity from IκBζ overexpression was confirmed by luciferase reporter assay. Finally, IκBζ coprecipitated with p65 and p50 NF-κB in NK cells in response to IL-12/IL-18, suggesting that IκBζ's effects on IFN-γ promoter activity are coregulated by NF-κB. These results suggest that IκBζ functions as an important regulator of IFN-γ in human NK cells, further expanding the class of IκBζ-modulated genes. PMID:21224476

  14. Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma

    PubMed Central

    Paoloni, Melissa; Mazcko, Christina; Selting, Kimberly; Lana, Susan; Barber, Lisa; Phillips, Jeffrey; Skorupski, Katherine; Vail, David; Wilson, Heather; Biller, Barbara; Avery, Anne; Kiupel, Matti; LeBlanc, Amy; Bernhardt, Anna; Brunkhorst, Beatrice; Tighe, Robert; Khanna, Chand

    2015-01-01

    Background Interleukin (IL)-12 is a pro-inflammatory cytokine that mediates T-helper type 1 responses and cytotoxic T-cell activation, contributing to its utility as anti-cancer agent. Systemic administration of IL-12 often results in unacceptable toxicity; therefore, strategies to direct delivery of IL-12 to tumors are under investigation. The objective of this study was to assist the preclinical development of NHS-IL12, an immunocytokine consisting of an antibody, which targets necrotic tumor regions, linked to IL-12. Specifically this study sought to evaluate the safety, serum pharmacokinetics, anti-tumor activity, and immune modulation of NHS-IL12 in dogs with naturally occurring cancers. Methodology/Principal Findings A rapid dose-escalation study of NHS-IL12 administered subcutaneously to dogs with melanoma was conducted through the Comparative Oncology Trials Consortium (COTC). Eleven dogs were enrolled in four dose-escalation cohorts; thereafter, an additional seven dogs were treated at the defined tolerable dose of 0.8 mg/m2. The expanded cohort at this fixed dose (ten dogs in total) was accrued for further pharmacokinetics and pharmacodynamics assessment. NHS-IL12 levels, serum cytokine concentrations, and peripheral blood mononuclear cell characterization (post-treatment) and draining lymph node immune profiling, and tumor biopsies (pre- and post-treatment) were collected. Adverse events included thrombocytopenia, liver enzymopathies, fever, and vasculitis. Correlation between interferon (IFN)-γ induction, adverse events, and NHS-IL12 exposure (maximum concentration and area under the concentration-time curve) were dose-dependent. Serum IL-10 levels and intratumoral CD8+ populations increased after treatment. Partial responses, according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were observed in two dogs treated with NHS-IL12 0.8 mg/m2 and 1.6 mg/m2. Conclusions/Significance NHS-IL12 was administered safely to dogs with melanoma

  15. Prevalence, Distribution and Functional Significance of the −237C to T Polymorphism in the IL-12Rβ2 Promoter in Indian Tuberculosis Patients

    PubMed Central

    Verma, Vikas Kumar; Taneja, Vibha; Jaiswal, Anand; Sharma, Sangeeta; Behera, Digamber; Sreenivas, Vishnubhatla; Chauhan, Shyam Singh; Prasad, Hanumanthappa Krishna

    2012-01-01

    Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the −237 polymorphic site in the 5′ promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 m

  16. Divergent signaling pathways regulate IL-12 production induced by different species of Lactobacilli in human dendritic cells.

    PubMed

    Amar, Yacine; Rizzello, Valeria; Cavaliere, Riccardo; Campana, Stefania; De Pasquale, Claudia; Barberi, Chiara; Oliveri, Daniela; Pezzino, Gaetana; Costa, Gregorio; Meddah, Aicha Tirtouil; Ferlazzo, Guido; Bonaccorsi, Irene

    2015-07-01

    Recent studies have indicated that different strains of Lactobacilli differ in their ability to regulate IL-12 production by dendritic cells (DCs), as some strains are stronger inducer of IL-12 while other are not and can even inhibit IL-12 production stimulated by IL-12-inducer Lactobacilli. In this report we demonstrate that Lactobacillus reuteri 5289, as previously described for other strains of L. reuteri, can inhibit DC production of IL-12 induced by Lactobacilllus acidophilus NCFM. Remarkably, L. reuteri 5289 was able to inhibit IL-12 production induced not only by Lactobacilli, as so far reported, but also by bacteria of different genera, including pathogens. We investigated in human DCs the signal transduction pathways involved in the inhibition of IL-12 production induced by L. reuteri 5289, showing that this potential anti-inflammatory activity, which is also accompanied by an elevated IL-10 production, is associated to a prolonged phosphorilation of ERK1/2 MAP kinase pathway. Improved understanding of the immune regulatory mechanisms exerted by Lactobacilli is crucial for a more precise employment of these commensal bacteria as probiotics in human immune-mediated pathologies, such as allergies or inflammatory bowel diseases.

  17. Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease.

    PubMed

    Sun, Lin; He, Chang; Nair, Lekha; Yeung, Justine; Egwuagu, Charles E

    2015-10-01

    Cytokines play crucial roles in coordinating the activities of innate and adaptive immune systems. In response to pathogen recognition, innate immune cells secrete cytokines that inform the adaptive immune system about the nature of the pathogen and instruct naïve T cells to differentiate into the appropriate T cell subtypes required to clear the infection. These include Interleukins, Interferons and other immune-regulatory cytokines that exhibit remarkable functional redundancy and pleiotropic effects. The focus of this review, however, is on the enigmatic Interleukin 12 (IL-12) family of cytokines. This family of cytokines plays crucial roles in shaping immune responses during antigen presentation and influence cell-fate decisions of differentiating naïve T cells. They also play essential roles in regulating functions of a variety of effector cells, making IL-12 family cytokines important therapeutic targets or agents in a number of inflammatory diseases, such as the CNS autoimmune diseases, uveitis and multiple sclerosis. Published by Elsevier Ltd.

  18. Diterpenes inhibit IL-12 production by DC and enhance Th2 cells polarization.

    PubMed

    Takei, Masao; Umeyama, Akemi; Arihara, Shigenobu

    2007-04-13

    Sugiol and 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (Secoferruginol) are diterpenes isolated from the heartwood of Cryptomeria japonica and are pharmacologically active substances. Dendritic cells (DC) have a key influence on the differentiation of naïve T cells into Th1 or Th2 effector cells. We demonstrate that Sugiol and Secoferruginol activate human DC as documented by phenotypic and functional maturation and altered cytokine production. Human monocytes were exposed to Sugiol or Secoferruginol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD83 on Sugiol-primed DC were enhanced. Sugiol dose-dependently inhibited IL-12p70 production by LPS-primed DC and to a lesser extent, the production of the proinflammatory cytokines. Naïve T cells co-cultured with Sugiol-primed DC, turned into typical Th2 which produced large quantities of IL-4 and released small amounts of IFN-gamma and reduced Th1 cell polarizing capacity. Sugiol-primed DC induced the development of Th2 cells via the enhanced expression of OX40L and augmented the Th2 cell polarizing capacity of DC via the inhibition of IL-12p70. Similar results were obtained with Secoferruginol. These results suggest that some diterpenes modulate human DC function in a fashion that favors Th2 cell polarization and might have implication in autoimmune diseases.

  19. IL-12/23p40-dependent clearance of Anaplasma phagocytophilum in the murine model of human anaplasmosis.

    PubMed

    Pedra, Joao H F; Tao, Jian; Sutterwala, Fayyaz S; Sukumaran, Bindu; Berliner, Nancy; Bockenstedt, Linda K; Flavell, Richard A; Yin, Zhinan; Fikrig, Erol

    2007-08-01

    Human anaplasmosis is an emerging infectious disease transmitted by ticks that can be potentially fatal in the immunocompromised and the elderly. The mechanisms of defense against the causative agent, Anaplasma phagocytophilum, are not completely understood; however, interferon (IFN)-gamma plays an important role in pathogen clearance. Here, we show that IFN-gamma is regulated through an early IL-12/23p40-dependent mechanism. Interleukin (IL)-12/23p40 is regulated in macrophages and dendritic cells after activation by microbial agonists and cytokines and constitutes a subunit of IL-12 and IL-23. IL-12/23p40-deficient mice displayed an increased A. phagocytophilum burden, accelerated thrombocytopenia and increased neutrophil numbers in the spleen at day 6 postinfection. Infection of MyD88- and mitogen-activated kinase kinase 3 (MKK3)-deficient mice suggested that the early susceptibility due to IL-12/23p40 deficiency was not dependent on signaling through MyD88 or MKK3. The lack of IL-12/23p40 reduced IFN-gamma production in both CD4(+) and CD8(+) T cells although the effect was more pronounced in CD4(+) T cells. Our data suggest that the immune response against A. phagocytophilum is a multifactorial and cooperative process. The IL-12/23p40 subunit drives the CD4(+) Th1 immune response in the early phase of infection and IL-12/23p40-independent mechanisms ultimately contribute to pathogen elimination from the host.

  20. An animal model for open femur fracture and osteomyelitis--Part II: Immunomodulation with systemic IL-12.

    PubMed

    Lindsey, Brock A; Clovis, Nina B; Smith, E Suzanne; Salihu, Sydha; Hubbard, David F

    2010-01-01

    Infection resulting from open fracture is a common problem in orthopedics. The purpose of this project was to study the effect of Interleukin-12 (IL-12) systemic therapy on a previously established open fracture model. One hundred seven male Sprague-Dawley rats were assigned to five groups: (1) normal (baseline), (2) control (controlled for anesthesia), (3) fracture, (4) staph, and (5) staph and IL-12 (SIL). Each group was divided into four time periods: 6, 10, 14, and 21 days after injury and fixation. The operative groups had a standardized femur fracture and fixation using a Kirschner wire as an intramedullary device. The two infection groups (staph and SIL) were inoculated with Staphylococcus aureus following fracture and fixed with an identical technique. The SIL group was treated with systemic IL-12 for a total of 10 doses over 10 days. Significantly decreased serum IL-12 levels were noted at day 10 in the operative groups compared to the normal and control groups. The SIL group showed significantly higher macrophage activation levels and total platelet counts at day 21 compared to all the other groups. The overall infection rate was not changed by IL-12 supplementation; however, bacterial qualitative growth scores were significantly lower in the SIL group at day 10, which corresponded to the lowest level of systemic IL-12 in the fracture group.

  1. Curcumin modulation of IFN-β and IL-12 signalling and cytokine induction in human T cells

    PubMed Central

    Fahey, Angela J; Adrian Robins, R; Constantinescu, Cris S

    2007-01-01

    Abstract Curcumin is a polyphenol derived from the dietary spice turmeric. It possesses diverse anti-inflammatory and anti-cancer properties. Curcumin has been shown to exhibit an inhibitory effect on the production of inflammatory cytokines by human monocytes and has inhibited the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in association with a decrease in interleukin 12 (IL-12) production and signal transducer and activator of transcription 4 (STAT4) activation. The type I interferon (IFN) IFN-has the ability to suppress IL-12. Both IL-12 and IFN-α/β signal through the activation by phosphorylation of STAT4. Our aim was to investigate the effects of curcumin on the ability of T cells to respond to IL-12 or IFN-α/β. We report that curcumin decreases IL-12-induced STAT4 phosphorylation, IFN-γ production, and IL-12 Rβ1 and β2 expression. IFN-β-induced STAT4 phosphorylation, IL-10 production and IFN receptor (IFNAR) subunits 1 and 2 expression were enhanced by curcumin. Curcumin increased IFN-α-induced IL-10 and IFNAR1 expression. Prior exposure to curcumin decreased IFN-α-induced IFNAR2 expression and did not modify the level of IFN-α-induced pSTAT4 generation. Thus, the effect of curcumin on STAT4 activation in T cells is dependent upon the stimulus to which the T cells have been exposed. PMID:17979888

  2. Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis.

    PubMed

    Becker, Christoph; Dornhoff, Heike; Neufert, Clemens; Fantini, Massimo C; Wirtz, Stefan; Huebner, Sabine; Nikolaev, Alexei; Lehr, Hans-Anton; Murphy, Andrew J; Valenzuela, David M; Yancopoulos, George D; Galle, Peter R; Karow, Margaret; Neurath, Markus F

    2006-09-01

    Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

  3. IL-12 enhances proliferation of peripheral blood mononuclear cells from Chagas' disease patients to Trypanosoma cruzi antigen.

    PubMed

    de Barros-Mazon, S; Guariento, M E; Abrahamsohn, I A

    1997-06-01

    Chagas' disease is caused by infection with Trypanosoma cruzi. Patients in the chronic phase of infection were grouped as belonging to the asymptomatic (or indeterminate), cardiac and cardiac plus digestive forms. Previous studies have described abnormal immune responsiveness by peripheral blood mononuclear cells (PBMC) from chronic chagasic patients. We report significant parasite antigen (T-Ag)-stimulated PBMC proliferative responses to be present in all three groups of patients. Treatment of T-Ag-stimulated cultures with rIL-12 significantly amplifies proliferative responses in all patients' groups, with similar rates of increment. IL-12 enhances T-Ag-specific lymphoproliferation without increasing proliferation of unstimulated PBMC from normal individuals or from patients. Comparatively, treatment with rIL-2 enhances both T-Ag-specific and unstimulated proliferation by PBMC from patients and normals. Thus, IL-12 acts on pre-activated cells while IL-2 also stimulates resting cells. No synergism was obtained by the combined use of IL-12 and IL-2. Therefore IL-12 can act as a more selective amplifier of T. cruzi reactive cells than IL-2. IL-12, by enhancing parasite-antigen specific immunity, could be of potential therapeutic use to control reactivated T. cruzi infections concomitant to AIDS or other situations of immunosuppression.

  4. Interleukin 12B (IL12B) Genetic Variation and Pulmonary Tuberculosis: A Study of Cohorts from The Gambia, Guinea-Bissau, United States and Argentina

    PubMed Central

    Hill, Philip C.; Wejse, Christian; Bisseye, Cyrille; Olesen, Rikke; Edwards, Todd L.; Gilbert, John R.; Myers, Jamie L.; Stryjewski, Martin E.; Abbate, Eduardo; Estevan, Rosa; Hamilton, Carol D.; Tacconelli, Alessandra; Novelli, Giuseppe; Brunetti, Ercole; Aaby, Peter; Sodemann, Morten; Østergaard, Lars; Adegbola, Richard; Williams, Scott M.; Scott, William K.; Sirugo, Giorgio

    2011-01-01

    We examined whether polymorphisms in interleukin-12B (IL12B) associate with susceptibility to pulmonary tuberculosis (PTB) in two West African populations (from The Gambia and Guinea-Bissau) and in two independent populations from North and South America. Nine polymorphisms (seven SNPs, one insertion/deletion, one microsatellite) were analyzed in 321 PTB cases and 346 controls from Guinea-Bissau and 280 PTB cases and 286 controls from The Gambia. For replication we studied 281 case and 179 control African-American samples and 221 cases and 144 controls of European ancestry from the US and Argentina. First-stage single locus analyses revealed signals of association at IL12B 3′ UTR SNP rs3212227 (unadjusted allelic p = 0.04; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61–0.99]) in Guinea-Bissau and rs11574790 (unadjusted allelic p = 0.05; additive genotypic p = 0.05, OR = 0.76, 95% CI [0.58–1.00]) in The Gambia. Association of rs3212227 was then replicated in African-Americans (rs3212227 allelic p = 0.002; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61–1.00]); most importantly, in the African-American cohort, multiple significant signals of association (seven of the nine polymorphisms tested) were detected throughout the gene. These data suggest that genetic variation in IL12B, a highly relevant candidate gene, is a risk factor for PTB in populations of African ancestry, although further studies will be required to confirm this association and identify the precise mechanism underlying it. PMID:21339808

  5. Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12

    PubMed Central

    Guo, Zhi; Gao, Hong-Yan; Zhang, Tian-Yang; Lou, Jin-Xing; Yang, Kai; Liu, Xiao-Dong; He, Xue-Peng; Chen, Hui-Ren

    2016-01-01

    The aim of the present study was to construct a chimeric adenovirus (Ad)5/F35 co-expressing human CD4O ligand (CD4OL) and interleukin (IL)-2 (Ad5/F35 CD40L-IL-2). The infection efficiency to human monocyte-derived dendritic cells (Mo-DCs), expression of genes, phenotype changes and IL-12 production of Mo-DC by Ad5/F35 CD40L-IL-2 were investigated. CD40L and IL-2 from total RNA extracted from human peripheral blood mononuclear cells (PBMCs) were cloned by reverse transcription-polymerase chain reaction and used to construct Ad5/F35 CD40L-IL-2. The infection efficiency, expression of CD40L, and phenotype changes of Mo-DC infected with Ad5/F35 CD40L-IL-2 were analyzed using flow cytometry. The quantities of IL-2 and IL-12 in the supernatants of Mo-DC following infection of Ad5/F35 CD40L-IL-2 were measured by enzyme-linked immunosorbent assay. The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed. Ad5/F35 CD40L-IL-2 efficiently infected Mo-DCs with an infection efficiency of >75%, and the infected Mo-DCs expressed CD40L and secreted IL-2. The expression levels of cluster of differentiation (CD)80, CD86, CD40, and human leukocyte antigen-antigen D related on Mo-DC were moderate; however, CD83 was low prior to infection of Ad5/F35 CD40L-IL-2. Those molecules, particularly CD83, were markedly upregulated 24 h after the infection. Increasing quantities of IL-12 in the supernatants were detected subsequent to infection at different time points in a time-dependent manner. Thus, Ad5/F35 CD40L-IL-2 efficiently infected human Mo-DCs and its products, CD40L and IL-2, were subsequently expressed. In addition, infection with Ad5/F35 CD40L-IL-2 stimulated the maturation of Mo-DC and high levels of IL-12 production. PMID:27882218

  6. Toxoplasma gondii-skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2

    PubMed Central

    2014-01-01

    Background The interest in the mechanisms involved in Toxoplasma gondii lipid acquisition has steadily increased during the past few decades, but it remains not completely understood. Here, we investigated the biogenesis and the fate of lipid droplets (LD) of skeletal muscle cells (SkMC) during their interaction with T. gondii by confocal and electron microscopy. We also evaluated whether infected SkMC modulates the production of prostaglandin E2 (PGE2), cytokines interleukin-12 (IL-12) and interferon-gamma (INF-g), and also the cyclooxygenase-2 (COX-2) gene induction. Methods Primary culture of skeletal muscle cells were infected with tachyzoites of T. gondii and analysed by confocal microscopy for observation of LD. Ultrastructural cytochemistry was also used for lipid and sarcoplasmatic reticulum (SR) detection. Dosage of cytokines (IL-12 and INF-g) by ELISA technique and enzyme-linked immunoassay (EIA) for PGE2 measurement were employed. The COX-2 gene expression analysis was performed by real time reverse transcriptase polymerase chain reaction (qRT-PCR). Results We demonstrated that T. gondii infection of SkMC leads to increase in LD number and area in a time course dependent manner. Moreover, the ultrastructural analysis demonstrated that SR and LD are in direct contact with parasitophorous vacuole membrane (PVM), within the vacuolar matrix, around it and interacting directly with the membrane of parasite, indicating that LD are recruited and deliver their content inside the parasitophorous vacuole (PV) in T. gondii-infected SkMC. We also observed a positive modulation of the production of IL-12 and IFN-g, increase of COX-2 mRNA levels in the first hour of T. gondii-SkMC interaction and an increase of prostaglandin E2 (PGE2) synthesis from 6 h up to 48 h of infection. Conclusions Taken together, the close association between SR and LD with PV could represent a source of lipids as well as other nutrients for the parasite survival, and together with the

  7. Revisiting human IL-12Rβ1 deficiency: a survey of 141 patients from 30 countries.

    PubMed

    de Beaucoudrey, Ludovic; Samarina, Arina; Bustamante, Jacinta; Cobat, Aurélie; Boisson-Dupuis, Stéphanie; Feinberg, Jacqueline; Al-Muhsen, Saleh; Jannière, Lucile; Rose, Yoann; de Suremain, Maylis; Kong, Xiao-Fei; Filipe-Santos, Orchidée; Chapgier, Ariane; Picard, Capucine; Fischer, Alain; Dogu, Figen; Ikinciogullari, Aydan; Tanir, Gonul; Al-Hajjar, Sami; Al-Jumaah, Suliman; Frayha, Husn H; AlSum, Zobaida; Al-Ajaji, Sulaiman; Alangari, Abdullah; Al-Ghonaium, Abdulaziz; Adimi, Parisa; Mansouri, Davood; Ben-Mustapha, Imen; Yancoski, Judith; Garty, Ben-Zion; Rodriguez-Gallego, Carlos; Caragol, Isabel; Kutukculer, Necil; Kumararatne, Dinakantha S; Patel, Smita; Doffinger, Rainer; Exley, Andrew; Jeppsson, Olle; Reichenbach, Janine; Nadal, David; Boyko, Yaryna; Pietrucha, Barbara; Anderson, Suzanne; Levin, Michael; Schandené, Liliane; Schepers, Kinda; Efira, André; Mascart, Françoise; Matsuoka, Masao; Sakai, Tatsunori; Siegrist, Claire-Anne; Frecerova, Klara; Blüetters-Sawatzki, Renate; Bernhöft, Jutta; Freihorst, Joachim; Baumann, Ulrich; Richter, Darko; Haerynck, Filomeen; De Baets, Frans; Novelli, Vas; Lammas, David; Vermylen, Christiane; Tuerlinckx, David; Nieuwhof, Chris; Pac, Malgorzata; Haas, Walther H; Müller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Levy, Jacob; Raj, Revathi; Cohen, Aileen Cleary; Lewis, David B; Holland, Steven M; Yang, Kuender D; Wang, Xiaochuan; Wang, Xiaohong; Jiang, Liping; Yang, Xiqiang; Zhu, Chaomin; Xie, Yuanyuan; Lee, Pamela Pui Wah; Chan, Koon Wing; Chen, Tong-Xin; Castro, Gabriela; Natera, Ivelisse; Codoceo, Ana; King, Alejandra; Bezrodnik, Liliana; Di Giovani, Daniela; Gaillard, Maria Isabel; de Moraes-Vasconcelos, Dewton; Grumach, Anete Sevciovic; da Silva Duarte, Alberto Jose; Aldana, Ruth; Espinosa-Rosales, Francisco Javier; Bejaoui, Mohammed; Bousfiha, Ahmed Aziz; Baghdadi, Jamila El; Ozbek, Namik; Aksu, Guzide; Keser, Melike; Somer, Ayper; Hatipoglu, Nevin; Aydogmus, Cigdem; Asilsoy, Suna; Camcioglu, Yildiz; Gülle, Saniye; Ozgur, Tuba T; Ozen, Meteran; Oleastro, Matias; Bernasconi, Andrea; Mamishi, Setareh; Parvaneh, Nima; Rosenzweig, Sergio; Barbouche, Ridha; Pedraza, Sigifredo; Lau, Yu Lung; Ehlayel, Mohammad S; Fieschi, Claire; Abel, Laurent; Sanal, Ozden; Casanova, Jean-Laurent

    2010-11-01

    Interleukin-12 receptor β1 (IL-12Rβ1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rβ1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought.

  8. IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression.

    PubMed

    Kanti Ghosh, Amlan; Sinha, Debolina; Mukherjee, Subhadeep; Biswas, Ratna; Biswas, Tapas

    2016-03-01

    Interleukin (IL)-15 is known to strongly modulate T-cell function; however, its role in controlling mucosal immunity, including its ability to modulate B-1a cell activity, remains to be elucidated. Here, we show that IL-15 upregulates activation molecules and the costimulatory molecule CD80 on viable B-1a cells. Cell activation was accompanied by the depletion of sialic acid-binding immunoglobulin-like lectin (Siglec)-G, an inhibitor of cell activation that is present on B-1a cells. The IL-15 receptor CD122 was stimulated on B-1a cells by the cytokine showing its direct involvement in IL-15-mediated responses. IL-10 is responsible for the long term survival of B-1a cells in culture, which is initially promoted by IL-15. The upregulation of IL-10 was followed by the appearance of suppressor of cytokine signaling (SOCS)1 in the presence of IL-15 and the loss of IL-10. This resulted in the cells switching to IL-12 expression. This anti-inflammatory to pro-inflammatory shift in the B-1a cell character was independent of the cell-specific marker CD5, which remained highly expressed throughout the in vitro life of the cells. The presence of the immunosuppressive receptor programmed cell death (PD)-1 and its ligand PD-L2 were features of a predominantly IL-10 response. PD-1 and PD-L2 can mediate juxtacrine signaling. However, the abrogation of PD-1 and its ligand was observed when the cells expressed IL-12. This demonstrates an inverse relationship between the receptor and ligand and the pro-inflammatory cytokine. The induction of IgM and IgA, which can play pivotal roles in mucosal immunity, was promoted in the presence of IL-15. Collectively, the data implicate IL-15 as the master cytokine that induces B-1a cells to mount a mucosal immune response.

  9. The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNalpha, TNFalpha and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60.

    PubMed

    Gil, S; Sepúlveda, N; Albina, E; Leitão, A; Martins, C

    2008-01-01

    The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNalpha, TNFalpha, IL12p40, TGFbeta and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNalpha, TNFalpha and IL12p40 expression was found in infection with NHV, in which expression of TGFbeta was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNalpha and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNalpha, TNFalpha and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection.

  10. An extrafollicular pathway for the generation of effector CD8+ T cells driven by the proinflammatory cytokine, IL-12

    PubMed Central

    Shah, Suhagi; Grotenbreg, Gijsbert M; Rivera, Amariliz; Yap, George S

    2015-01-01

    The proinflammatory cytokine IL-12 drives the generation of terminally differentiated KLRG1+ effector CD8+ T cells. Using a Toxoplasma vaccination model, we delineate the sequence of events that naïve CD8+ T cells undergo to become terminal effectors and the differentiation steps controlled by IL-12. We demonstrate that direct IL-12 signaling on CD8+ T cells is essential for the induction of KLRG1 and IFN-γ, but the subsequent downregulation of CXCR3 is controlled by IL-12 indirectly through the actions of IFN-γ and IFN-γ-inducible chemokines. Differentiation of nascent effectors occurs in an extrafollicular splenic compartment and is driven by late IL-12 production by DCs distinct from the classical CD8α+ DC. Unexpectedly, we also found extensive proliferation of both KLRG1− and KLRG1+ CD8+ T cells in the marginal zone and red pulp, which ceases prior to the final KLRG1Hi CXCR3Lo stage. Our findings highlight the notion of an extrafollicular pathway for effector T cell generation. DOI: http://dx.doi.org/10.7554/eLife.09017.001 PMID:26244629

  11. Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFNgamma production.

    PubMed

    Lemos, Henrique P; Grespan, Renata; Vieira, Silvio M; Cunha, Thiago M; Verri, Waldiceu A; Fernandes, Karla S S; Souto, Fabricio O; McInnes, Iain B; Ferreira, Sergio H; Liew, Foo Y; Cunha, Fernando Q

    2009-04-07

    IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN gamma production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFNgamma but was enhanced by prostaglandin E(2) (PGE(2)). IL-23-induced IL-17 production was increased by PGE(2) and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFNgamma-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFNgamma but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNFalpha, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFNgamma production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

  12. Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFNγ production

    PubMed Central

    Lemos, Henrique P.; Grespan, Renata; Vieira, Silvio M.; Cunha, Thiago M.; Verri, Waldiceu A.; Fernandes, Karla S. S.; Souto, Fabricio O.; McInnes, Iain B.; Ferreira, Sergio H.; Liew, Foo Y.; Cunha, Fernando Q.

    2009-01-01

    IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN γ production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFNγ but was enhanced by prostaglandin E2 (PGE2). IL-23-induced IL-17 production was increased by PGE2 and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFNγ-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFNγ but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNFα, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFNγ production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs. PMID:19289819

  13. Enhanced expression of IL-10 in contrast to IL-12B mRNA in poultry with experimental coccidiosis.

    PubMed

    Haritova, A M; Stanilova, S A

    2012-11-01

    Interleukin (IL)-12 and IL-10 are immunoregulatory cytokines with an antagonistic effect on the T-helper (Th)1/Th2 cytokine balance and they provide a functional link between innate resistance and the adaptive immune response. This investigation was conducted to determine the expression of IL-10 and IL-12B mRNA levels in chickens' gut mucosa infected with Eimeria tenella and in sulfachlorpyrazine-sodium treated animals after infection. Broiler chickens were randomly allocated in three groups: healthy untreated control; infected untreated animals and infected, treated with sulfachlorpyrazine sodium chickens 6 days after the challenge with an E. tenella. Quantitative real time PCR analysis was performed using specific primer pairs and probes for IL-10 and IL-12B. The expression of IL-10 mRNA was greater in the duodenum then in the caecum and the liver of healthy chickens. E. tenella infection led to significant up-regulation of IL-10 mRNA in the caecum, followed by mRNA in the liver. A significant down regulation was observed mainly in the caecum after the treatment with sulfachlorpyrazine. In contrast, IL-12B expression in all investigated tissues remained insignificantly affected in the studied groups of animals. Distinct up-regulation of IL-10 mRNA, after the challenge with E. tenella, in the caecum can be attributed to the tissue tropism of Eimeria spp. The production of IL-12 is regulated by negative feedback through IL-10 which explains lack of increase in IL-12B mRNA. Sulfonamide treatment resulted in clinical improvement and restoration of IL-10 mRNA to the levels observed in healthy chickens. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Evaluation in macaques of HIV-1 DNA vaccines containing primate CpG motifs and fowlpoxvirus vaccines co-expressing IFNgamma or IL-12.

    PubMed

    Dale, C Jane; De Rose, Robert; Wilson, Kim M; Croom, Hayley A; Thomson, Scott; Coupar, Barbara E H; Ramsay, Alistair; Purcell, Damian F J; Ffrench, Rosemary; Law, Matthew; Emery, Sean; Cooper, David A; Ramshaw, Ian A; Boyle, David B; Kent, Stephen J

    2004-11-25

    Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV. Plasmid DNA vaccines and recombinant fowlpoxvirus (rFPV) vaccines are promising HIV-1 vaccine candidates, although either vaccine alone may be insufficient to protect against HIV-1. A consecutive immunisation strategy involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV-1 antigens was further evaluated in 30 macaques. The DNA vaccine vector included CpG immunostimulatory molecules, and rFPV vaccines were compared with rFPV vaccines co-expressing the pro-T cell cytokines IFNgamma or IL-12. Vaccines expressed multiple HIV-1 genes, mutated to remove active sites of the HIV proteins. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1 specific T lymphocyte responses was observed following rFPV boosting. Co-expression of IFNgamma or IL-12 by the rFPV vaccines did not further enhance immune responses. Non-sterilising protection from a non-pathogenic HIV-1 challenge was observed. This study provides evidence of a safe, optimised, strategy for the generation of T-cell mediated immunity to HIV-1.

  15. MCP1 SNPs and Pulmonary Tuberculosis in Cohorts from West Africa, the USA and Argentina: Lack of Association or Epistasis with IL12B Polymorphisms

    PubMed Central

    Velez Edwards, Digna R.; Tacconelli, Alessandra; Wejse, Christian; Hill, Philip C.; Morris, Gerard A. J.; Edwards, Todd L.; Gilbert, John R.; Myers, Jamie L.; Park, Yo Son; Stryjewski, Martin E.; Abbate, Eduardo; Estevan, Rosa; Rabna, Paulo; Novelli, Giuseppe; Hamilton, Carol D.; Adegbola, Richard; Østergaard, Lars; Williams, Scott M.; Scott, William K.; Sirugo, Giorgio

    2012-01-01

    The monocyte chemotactic protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of monocytes to M. tuberculosis infection sites, and previous studies have reported that genetic variants in MCP1 are associated with differential susceptibility to pulmonary tuberculosis (PTB). We examined eight MCP1 single nucleotide polymorphisms (SNPs) in a multi-ethnic, case-control design that included: 321 cases and 346 controls from Guinea-Bissau, 258 cases and 271 controls from The Gambia, 295 cases and 179 controls from the U.S. (African-Americans), and an additional set of 237 cases and 144 controls of European ancestry from the U.S. and Argentina. Two locus interactions were also examined for polymorphisms in MCP1 and interleukin 12B (IL12B), another gene implicated in PTB risk. Examination of previously associated MCP1 SNPs rs1024611 (−2581A/G), rs2857656 (−362G/C) and rs4586 (+900C/T) did not show evidence for association. One interaction between rs2857656 and IL12B SNP rs2288831 was observed among Africans but the effect was in the opposite direction in Guineans (OR = 1.90, p = 0.001) and Gambians (OR = 0.64, p = 0.024). Our data indicate that the effect of genetic variation within MCP1 is not clear cut and additional studies will be needed to elucidate its role in TB susceptibility. PMID:22384203

  16. Safety and Immunogenicity of an HIV-1 Gag DNA Vaccine with or without IL-12 and/or IL-15 Plasmid Cytokine Adjuvant in Healthy, HIV-1 Uninfected Adults

    PubMed Central

    Kalams, Spyros A.; Parker, Scott; Jin, Xia; Elizaga, Marnie; Metch, Barbara; Wang, Maggie; Hural, John; Lubeck, Michael; Eldridge, John; Cardinali, Massimo; Blattner, William A.; Sobieszczyk, Magda; Suriyanon, Vinai; Kalichman, Artur; Weiner, David B.; Baden, Lindsey R.

    2012-01-01

    Background DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques. Methodology/Principal Findings We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response. Conclusions/Significance This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity. Trial Registration Clinicaltrials.gov NCT00115960 NCT00111605 PMID:22242162

  17. Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism.

    PubMed

    Vacaflores, Aldo; Chapman, Nicole M; Harty, John T; Richer, Martin J; Houtman, Jon C D

    2016-01-01

    Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism.

  18. Abrogation of TNFα Production during Cancer Immunotherapy Is Crucial for Suppressing Side Effects Due to the Systemic Expression of IL-12

    PubMed Central

    Barrios, Bibiana; Baez, Natalia S.; Reynolds, Della; Iribarren, Pablo; Cejas, Hugo; Young, Howard A.; Rodriguez-Galan, Maria Cecilia

    2014-01-01

    For more than a decade, the cytokine interleukin-12 (IL-12) has been utilized, either alone or in combination with other drugs, as a treatment for cancer. The numerous anti-tumor properties of IL-12 still generate interest in the clinical use of this cytokine, even though it has demonstrated toxicity when administrated systemically. As an approach to overcome this toxicity, numerous laboratories have attempted to induce IL-12 expression at the site of the tumor. However for tumors that are difficult to remove surgically or for the treatment of disseminated metastases, systemic expression of this cytokine still remains as the most efficient method of administration. Nevertheless, finding alternative approaches for the use of IL-12 in the treatment of cancer and unraveling the basis of IL-12-side effects remain a challenge. In the present work we demonstrate that systemic expression of IL-12 through hydrodynamic injection of IL-12 cDNA is able to induce different types of liver lesions associated with a toxic pathology. However we report here that hepatic toxicity is diminished and survival of mice enhanced in the absence of tumor necrosis factor alpha (TNFα). This observation is in contrast to several murine models and clinical trials that postulate interferon gamma (IFNγ) as the main cytokine responsible for IL-12 toxicity. Moreover, our work demonstrates that when IL-12 cDNA is co-injected with IL-18 cDNA or when mice are pre-treated with a low dose of IL-12 cDNA prior to receiving a high dose of IL-12 cDNA, systemic levels of TNFα are almost completely abrogated, resulting in improved survival and less hepatic damage. Importantly, abrogation of TNFα signaling does not affect the strong anti-tumor activity of IL-12. Thus, neutralizing TNFα with antagonists already approved for human use offers a promising approach to abrogate IL-12 side effects during the use of this cytokine for the treatment of cancer. PMID:24587231

  19. Abrogation of TNFα production during cancer immunotherapy is crucial for suppressing side effects due to the systemic expression of IL-12.

    PubMed

    Barrios, Bibiana; Baez, Natalia S; Reynolds, Della; Iribarren, Pablo; Cejas, Hugo; Young, Howard A; Rodriguez-Galan, Maria Cecilia

    2014-01-01

    For more than a decade, the cytokine interleukin-12 (IL-12) has been utilized, either alone or in combination with other drugs, as a treatment for cancer. The numerous anti-tumor properties of IL-12 still generate interest in the clinical use of this cytokine, even though it has demonstrated toxicity when administrated systemically. As an approach to overcome this toxicity, numerous laboratories have attempted to induce IL-12 expression at the site of the tumor. However for tumors that are difficult to remove surgically or for the treatment of disseminated metastases, systemic expression of this cytokine still remains as the most efficient method of administration. Nevertheless, finding alternative approaches for the use of IL-12 in the treatment of cancer and unraveling the basis of IL-12-side effects remain a challenge. In the present work we demonstrate that systemic expression of IL-12 through hydrodynamic injection of IL-12 cDNA is able to induce different types of liver lesions associated with a toxic pathology. However we report here that hepatic toxicity is diminished and survival of mice enhanced in the absence of tumor necrosis factor alpha (TNFα). This observation is in contrast to several murine models and clinical trials that postulate interferon gamma (IFNγ) as the main cytokine responsible for IL-12 toxicity. Moreover, our work demonstrates that when IL-12 cDNA is co-injected with IL-18 cDNA or when mice are pre-treated with a low dose of IL-12 cDNA prior to receiving a high dose of IL-12 cDNA, systemic levels of TNFα are almost completely abrogated, resulting in improved survival and less hepatic damage. Importantly, abrogation of TNFα signaling does not affect the strong anti-tumor activity of IL-12. Thus, neutralizing TNFα with antagonists already approved for human use offers a promising approach to abrogate IL-12 side effects during the use of this cytokine for the treatment of cancer.

  20. Genetic characterization of interleukins (IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphs

    PubMed Central

    Neves, Fabiana; Abrantes, Joana; Almeida, Tereza; de Matos, Ana Lemos; Costa, Paulo P

    2015-01-01

    ILs, as essential innate immune modulators, are involved in an array of biological processes. In the European rabbit (Oryctolagus cuniculus) IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18 have been implicated in inflammatory processes and in the immune response against rabbit hemorrhagic disease virus and myxoma virus infections. In this study we characterized these ILs in six Lagomorpha species (European rabbit, pygmy rabbit, two cottontail rabbit species, European brown hare and American pika). Overall, these ILs are conserved between lagomorphs, including in their exon/intron structure. Most differences were observed between leporids and American pika. Indeed, when comparing both, some relevant differences were observed in American pika, such as the location of the stop codon in IL-1α and IL-2, the existence of a different transcript in IL8 and the number of cysteine residues in IL-1β. Changes at N-glycosylation motifs were also detected in IL-1, IL-10, IL-12B and IL-15. IL-1α is the protein that presents the highest evolutionary distances, which is in contrast to IL-12A where the distances between lagomorphs are the lowest. For all these ILs, sequences of human and European rabbit are more closely related than between human and mouse or European rabbit and mouse. PMID:26395994

  1. Genetic characterization of interleukins (IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphs.

    PubMed

    Neves, Fabiana; Abrantes, Joana; Almeida, Tereza; de Matos, Ana Lemos; Costa, Paulo P; Esteves, Pedro J

    2015-11-01

    ILs, as essential innate immune modulators, are involved in an array of biological processes. In the European rabbit (Oryctolagus cuniculus) IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18 have been implicated in inflammatory processes and in the immune response against rabbit hemorrhagic disease virus and myxoma virus infections. In this study we characterized these ILs in six Lagomorpha species (European rabbit, pygmy rabbit, two cottontail rabbit species, European brown hare and American pika). Overall, these ILs are conserved between lagomorphs, including in their exon/intron structure. Most differences were observed between leporids and American pika. Indeed, when comparing both, some relevant differences were observed in American pika, such as the location of the stop codon in IL-1α and IL-2, the existence of a different transcript in IL8 and the number of cysteine residues in IL-1β. Changes at N-glycosylation motifs were also detected in IL-1, IL-10, IL-12B and IL-15. IL-1α is the protein that presents the highest evolutionary distances, which is in contrast to IL-12A where the distances between lagomorphs are the lowest. For all these ILs, sequences of human and European rabbit are more closely related than between human and mouse or European rabbit and mouse. © The Author(s) 2015.

  2. Early infiltration of p40IL12+CCR7+CD11b+ cells is critical for fibrosis development

    PubMed Central

    Correa‐Costa, Matheus; Azevedo, Hatylas; Silva, Reinaldo Correia; Cruz, Mario Costa; Almeida, Maira Estanislau Soares; Hiyane, Meire Ioshie; Moreira‐Filho, Carlos Alberto; Santos, Marinilce Fagundes; Perez, Katia Regina; Cuccovia, Iolanda Midea; Camara, Niels Olsen Saraiva

    2016-01-01

    Abstract Introduction Macrophages are heterogeneous and thus can be correlated with distinct tissue outcomes after injury. Conflicting data have indicated that the M2‐related phenotype directly triggers fibrosis. Conversely, we hypothesize here that the inflammatory milieu provided by early infiltration of pro‐inflammatory macrophages dictates tissue scarring after injury. Methods and Results We first determined that tissue‐localized macrophages exhibit a pro‐inflammatory phenotype (p40IL12+CCR7+CD11b+) during the early phase of a chronic injury model, in contrast to a pro‐resolving phenotype (Arg1+IL10+CD206+CD11b+) at a later stage. Then, we evaluated the effects of injecting macrophages differentiated in vitro in the presence of IFNγ + LPS or IL4 + IL13 or non‐differentiated macrophages (hereafter, M0) on promoting inflammation and progression of chronic injury in macrophage‐depleted mice. In addition to enhancing the expression of pro‐inflammatory cytokines, the injection of M (IFNγ + LPS), but not M (IL4 + IL13) or M0, accentuated fibrosis while augmenting levels of anti‐inflammatory molecules, increasing collagen deposition and impairing organ function. We observed a similar profile after injection of sorted CCR7+CD11b+ cells and a more pronounced effect of M (IFNγ + LPS) cells originated from Stat6−/− mice. The injection of M (IFNγ + LPS) cells was associated with the up‐regulation of inflammation‐ and fibrosis‐related proteins (Thbs1, Mmp7, Mmp8, and Mmp13). Conclusions Our results suggest that pro‐inflammatory macrophages promote microenvironmental changes that may lead to fibrogenesis by inducing an inflammatory milieu that alters a network of extracellular‐related genes, culminating in tissue fibrosis. PMID:27621813

  3. Thimerosal compromises human dendritic cell maturation, IL-12 production, chemokine release, and T-helper polarization.

    PubMed

    Loison, Emily; Gougeon, Marie-Lise

    2014-01-01

    Thimerosal is a preservative used in multidose vials of vaccine formulations to prevent bacterial and fungal contamination. We recently reported that nanomolar concentrations of thimerosal induce cell cycle arrest of human T cells activated via the TCR and inhibition of proinflammatory cytokine production, thus interfering with T-cell functions. Given the essential role of dendritic cells (DCs) in T-cell polarization and vaccine immunity, we studied the influence of non-toxic concentrations of thimerosal on DC maturation and functions. Ex-vivo exposure of human monocyte-derived DCs to nanomolar concentrations of thimerosal prevented LPS-induced DC maturation, as evidenced by the inhibition of morphological changes and a decreased expression of the maturation markers CD86 and HLA-DR. In addition thimerosal dampened their proinflammatory response, in particular the production of the Th1 polarizing cytokine IL-12, as well as TNF-α and IL-6. DC-dependent T helper polarization was altered, leading to a decreased production of IFN-γ IP10 and GM-CSF and increased levels of IL-8, IL-9, and MIP-1α. Although multi-dose vials of vaccines containing thimerosal remain important for vaccine delivery, our results alert about the ex-vivo immunomodulatory effects of thimerosal on DCs, a key player for the induction of an adaptive response.

  4. Thimerosal compromises human dendritic cell maturation, IL-12 production, chemokine release, and T-helper polarization

    PubMed Central

    Loison, Emily; Gougeon, Marie-Lise

    2014-01-01

    Thimerosal is a preservative used in multidose vials of vaccine formulations to prevent bacterial and fungal contamination. We recently reported that nanomolar concentrations of thimerosal induce cell cycle arrest of human T cells activated via the TCR and inhibition of proinflammatory cytokine production, thus interfering with T-cell functions. Given the essential role of dendritic cells (DCs) in T-cell polarization and vaccine immunity, we studied the influence of non-toxic concentrations of thimerosal on DC maturation and functions. Ex-vivo exposure of human monocyte-derived DCs to nanomolar concentrations of thimerosal prevented LPS-induced DC maturation, as evidenced by the inhibition of morphological changes and a decreased expression of the maturation markers CD86 and HLA-DR. In addition thimerosal dampened their proinflammatory response, in particular the production of the Th1 polarizing cytokine IL-12, as well as TNF-α and IL-6. DC-dependent T helper polarization was altered, leading to a decreased production of IFN-γ IP10 and GM-CSF and increased levels of IL-8, IL-9, and MIP-1α. Although multi-dose vials of vaccines containing thimerosal remain important for vaccine delivery, our results alert about the ex-vivo immunomodulatory effects of thimerosal on DCs, a key player for the induction of an adaptive response PMID:25424939

  5. NOD Dendritic Cells Stimulated with Lactobacilli Preferentially Produce IL-10 versus IL-12 and Decrease Diabetes Incidence

    PubMed Central

    Manirarora, Jean N.; Parnell, Sarah A.; Hu, Yoon-Hyeon; Kosiewicz, Michele M.; Alard, Pascale

    2011-01-01

    Dendritic cells (DCs) from NOD mice produced high levels of IL-12 that induce IFNγ-producing T cells involved in diabetes development. We propose to utilize the microorganism ability to induce tolerogenic DCs to abrogate the proinflammatory process and prevent diabetes development. NOD DCs were stimulated with Lactobacilli (nonpathogenic bacteria targeting TLR2) or lipoteichoic acid (LTA) from Staphylococcus aureus (TLR2 agonist). LTA-treated DCs produced much more IL-12 than IL-10 and accelerated diabetes development when transferred into NOD mice. In contrast, stimulation of NOD DCs with L. casei favored the production of IL-10 over IL-12, and their transfer decreased disease incidence which anti-IL-10R antibodies restored. These data indicated that L. casei can induce NOD DCs to develop a more tolerogenic phenotype via production of the anti-inflammatory cytokine, IL-10. Evaluation of the relative production of IL-10 and IL-12 by DCs may be a very useful means of identifying agents that have therapeutic potential. PMID:21716731

  6. NK-Cell Recruitment Is Necessary for Eradication of Peritoneal Carcinomatosis with an IL12-Expressing Maraba Virus Cellular Vaccine.

    PubMed

    Alkayyal, Almohanad A; Tai, Lee-Hwa; Kennedy, Michael A; de Souza, Christiano Tanese; Zhang, Jiqing; Lefebvre, Charles; Sahi, Shalini; Ananth, Abhirami A; Mahmoud, Ahmad Bakur; Makrigiannis, Andrew P; Cron, Greg O; Macdonald, Blair; Marginean, E Celia; Stojdl, David F; Bell, John C; Auer, Rebecca C

    2017-03-01

    Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death from abdominal cancers. Immunotherapies have been effective for selected solid malignancies, but their potential in PC has been little explored. Here, we report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex vivo with an oncolytic Maraba MG1 virus expressing IL12, promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of IFNγ-induced protein-10 (IP-10) from dendritic cells. The recruitment of cytotoxic, IFNγ-secreting NK cells was associated with reduced tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors > 8 mm), a complete radiologic response was demonstrated within 8 to14 weeks, associated with 100% long-term survival. The impact of MG1-IL12-ICV upon NK-cell recruitment and function observed in the murine system was recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. These findings suggest that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year. Cancer Immunol Res; 5(3); 211-21. ©2017 AACR. ©2017 American Association for Cancer Research.

  7. Severe Enteropathy and Hypogammaglobulinemia Complicating Refractory Mycobacterium tuberculosis Complex Disseminated Disease in a Child with IL-12Rβ1 Deficiency.

    PubMed

    Arias, Andrés Augusto; Perez-Velez, Carlos M; Orrego, Julio César; Moncada-Velez, Marcela; Rojas, Jessica Lineth; Wilches, Alejandra; Restrepo, Andrea; Trujillo, Mónica; Garcés, Carlos; Arango-Ferreira, Catalina; González, Natalia; Oleaga-Quintas, Carmen; Fernández, Diana; Isaza-Correa, Johana Marcela; Gongóra, Diego Eduardo; Gonzalez-Loaiza, Daniel; Sierra, Juan Esteban; Casanova, Jean Laurent; Bustamante, Jacinta; Franco, José Luis

    2017-09-01

    Mendelian susceptibility to mycobacterial disease is a rare clinical condition characterized by a predisposition to infectious diseases caused by poorly virulent mycobacteria. Other infections such as salmonellosis and candidiasis are also reported. The purpose of this article is to describe a young boy affected with various infectious diseases caused by Mycobacterium tuberculosis complex, Salmonella sp, Klebsiella pneumonie, Citrobacter sp., and Candida sp, complicated with severe enteropathy and transient hypogammaglobulinemia. We reviewed medical records and performed flow cytometry staining for lymphocyte populations, lymphocyte proliferation in response to PHA, and intracellular IFN-γ production in T cell PHA blasts in the patient and a healthy control. Sanger sequencing was used to confirm the genetic variants in the patient and relatives. Genetic analysis revealed a bi-allelic mutation in IL12RB1 (C291Y) resulting in complete IL-12Rβ1 deficiency. Functional analysis demonstrated the lack of intracellular production of IFN-γ in CD3+ T lymphocytes from the patient in response to rhIL-12p70. To our knowledge, this is the third patient with MSMD due to IL-12Rβ1 deficiency complicated with enteropathy and hypogammaglobulinemia and the first case of this disease to be described in Colombia.

  8. Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages.

    PubMed

    Kanemaru, Hisashi; Yamane, Fumihiro; Fukushima, Kiyoharu; Matsuki, Takanori; Kawasaki, Takahiro; Ebina, Isao; Kuniyoshi, Kanako; Tanaka, Hiroki; Maruyama, Kenta; Maeda, Kazuhiko; Satoh, Takashi; Akira, Shizuo

    2017-08-29

    The development of effective treatments against cancers is urgently needed, and the accumulation of CD8(+) T cells within tumors is especially important for cancer prognosis. Although their mechanisms are still largely unknown, growing evidence has indicated that innate immune cells have important effects on cancer progression through the production of various cytokines. Here, we found that basic leucine zipper transcription factor ATF-like 2 (Batf2) has an antitumor effect. An s.c. inoculated tumor model produced fewer IL-12 p40(+) macrophages and activated CD8(+) T cells within the tumors of Batf2(-/-) mice compared with WT mice. In vitro studies also revealed that the IL-12 p40 expression was significantly lower in Batf2(-/-) macrophages following their stimulation by toll-like receptor ligands, such as R848. Additionally, we found that BATF2 interacts with p50/p65 and promotes IL-12 p40 expression. In conclusion, Batf2 has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces CD8(+) T-cell activation and accumulation within the tumor.

  9. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

    PubMed

    Osada, Takuya; Berglund, Peter; Morse, Michael A; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Yang, Xiao Yi; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R; Clay, Timothy M; Smith, Jonathan; Kim Lyerly, H

    2012-11-01

    We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

  10. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and anti-tumor effects

    PubMed Central

    Osada, Takuya; Berglund, Peter; Morse, Michael A.; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2013-01-01

    We recently demonstrated that Venezuelan equine encephalitis (VEE) virus-based replicon particles (VRP) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP expressing Interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and anti-tumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)) and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12 and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing anti-tumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted. PMID:22488274

  11. Therapeutic and prophylactic thalidomide in TNBS-induced colitis: Synergistic effects on TNF-α, IL-12 and VEGF production

    PubMed Central

    Carvalho, Ana Teresa; Souza, Heitor; Carneiro, Antonio Jose; Castelo-Branco, Morgana; Madi, Kalil; Schanaider, Alberto; Silva, Flavia; Pereira Jứnior, Fernando Antonio; Pereira, Márcia G; Tortori, Cláudio; Dines, Ilana; Carvalho, Jane; Rocha, Eduardo; Elia, Celeste

    2007-01-01

    AIM: To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor α (TNF-α), IL-12, and vascular endothelial growth factor (VEGF), hallmarks of intestinal inflammation in Crohn’s disease (CD). METHODS: Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4+ T cells, macrophages, and VEGF+ cells were detected by immunohistochemistry. TNF-α and IL-12 were quantified in the supernatant of organ cultures by ELISA. RESULTS: Significant reduction in the inflammatory score and in the percentage of VEGF+ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-α and IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide. TNF-α levels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-α and IL-12 were significantly correlated with the inflammatory score and the number of VEGF+ cells. CONCLUSION: Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-α, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD. PMID:17465495

  12. IL-12-based vaccination therapy reverses liver-induced systemic tolerance in a mouse model of hepatitis B virus carrier.

    PubMed

    Zeng, Zhutian; Kong, Xiaohui; Li, Fenglei; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2013-10-15

    Liver-induced systemic immune tolerance that occurs during chronic hepadnavirus infection is the biggest obstacle for effective viral clearance. Immunotherapeutic reversal of this tolerance is a promising strategy in the clinic but remains to be explored. In this study, using a hepatitis B virus (HBV)-carrier mouse model, we report that IL-12-based vaccination therapy can efficiently reverse systemic tolerance toward HBV. HBV-carrier mice lost responsiveness to hepatitis B surface Ag (HBsAg) vaccination, and IL-12 alone could not reverse this liver-induced immune tolerance. However, after IL-12-based vaccination therapy, the majority of treated mice became HBsAg(-) in serum; hepatitis B core Ag was also undetectable in hepatocytes. HBV clearance was dependent on HBsAg vaccine-induced anti-HBV immunity. Further results showed that IL-12-based vaccination therapy strongly enhanced hepatic HBV-specific CD8(+) T cell responses, including proliferation and IFN-γ secretion. Systemic HBV-specific CD4(+) T cell responses were also restored in HBV-carrier mice, leading to the arousal of HBsAg-specific follicular Th-germinal center B cell responses and anti-hepatitis B surface Ag Ab production. Recovery of HBsAg-specific responses also correlated with both reduced CD4(+)Foxp3(+) regulatory T cell frequency and an enhanced capacity of effector T cells to overcome inhibition by regulatory T cells. In conclusion, IL-12-based vaccination therapy may reverse liver-induced immune tolerance toward HBV by restoring systemic HBV-specific CD4(+) T cell responses, eliciting robust hepatic HBV-specific CD8(+) T cell responses, and facilitating the generation of HBsAg-specific humoral immunity; thus, this therapy may become a viable approach to treating patients with chronic hepatitis B.

  13. Up-regulation of IL-12 expression in patients with chronic hepatitis B is mediated by the PI3K/Akt pathway.

    PubMed

    Wang, Hong-wei; Gao, Hai-li; Wei, Xiao-xia; Wang, Xin-hong

    2015-09-01

    Hepatitis B virus (HBV) replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes.IL-12 is a typical proinflammatory cytokine that plays a critical role in host defense against pathogens, including the HBV. However, the role of IL-12 in chronic hepatitis B (CHB) remains unclear. The aims of this study were to detect the expression of IL-12 in CHB patients and explore the molecular mechanism of HBV-induced IL-12 expression. The results showed that serum levels and hepatic expression of IL-12 were significantly upregulated in CHB patients. HBx protein increased IL-12 expression in a dose-dependent manner. Furthermore, inhibition of PI3K/Akt significantly decreased the HBx-induced IL-12 expression and Akt activation. Taken together, these results indicate that the molecular mechanism of HBV-induced IL-12 expression involves activation of the PI3K/Akt pathway by HBx, leading to transactivation of the IL-12 p35 and p40 promoters.

  14. Opposing Roles for CXCR3 Signaling in Central Nervous System Versus Ocular Inflammation Mediated by the Astrocyte-Targeted Production of IL-12

    PubMed Central

    Krauthausen, Marius; Ellis, Sally L.; Zimmermann, Julian; Sarris, Maria; Wakefield, Denis; Heneka, Michael T.; Campbell, Iain L.; Müller, Marcus

    2011-01-01

    CXCR3 and its ligands are important for the trafficking of activated CD4+ TH1 T cells, CD8+ T cells, and natural killer cells during inflammation. Recent functional studies demonstrate a more diverse role of CXCR3 in inflammatory diseases of the central nervous system (CNS). We examined the impact of CXCR3 on a less complex interferon-γ–dependent, type 1 cell–mediated immune response in the CNS, induced in mice by the transgenic production of glial fibrillary acidic protein IL-12 (GF-IL12) by astrocytes and retinal Müller cells. GF-IL12 mice develop ataxia because of severe cerebellar inflammation but have little overt ocular disease. Surprisingly, CXCR3-deficient GF-IL12 mice (GF-IL12/CXCR3KO) have drastically reduced ataxia but developed cataracts, severe ocular inflammation, and eye atrophy. Most GF-IL12/CXCR3KO mice had minimal cerebellar inflammation but severe retinal disorganization, loss of photoreceptors, and lens destruction in the eye. The number of CD3+, CD11b+, and natural killer 1.1+ cells were reduced in the CNS but highly increased in the eyes of GF-IL12/CXCR3KO compared with GF-IL12 mice. High levels of interferon-γ, IL-1, tumor necrosis factor α, CXCL9, CXCL10, and CCL5 were found in GF-IL12 cerebelli and GF-IL12/CXCR3KO eyes. Our findings demonstrate key but paradoxical functions for CXCR3 in IL-12–induced immune disease in the CNS, promoting inflammation in the brain yet restricting it in the eye. We conclude that the function of CXCR3 in cellular immune disease is driven by a common trigger and is controlled by tissue-specific factors. PMID:21925471

  15. Enhanced antitumor effects by combining an IL-12/anti-DNA fusion protein with avelumab, an anti-PD-L1 antibody.

    PubMed

    Fallon, Jonathan K; Vandeveer, Amanda J; Schlom, Jeffrey; Greiner, John W

    2017-03-28

    The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. It is shown here that NHS-muIL12, in an IFN-γ‒dependent mechanism, upregulates mPD-L1 expression on mouse tumors, which could be construed as an immunosuppressive action. Yet concurrent therapy with NHS-muIL12 and an anti-PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1‒expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies.

  16. Serum IL-12 Is Increased in Mexican Obese Subjects and Associated with Low-Grade Inflammation and Obesity-Related Parameters

    PubMed Central

    Suárez-Álvarez, K.; Solís-Lozano, L.; Leon-Cabrera, S.; González-Chávez, A.; Gómez-Hernández, G.; Quiñones-Álvarez, M. S.; Serralde-Zúñiga, A. E.; Hernández-Ruiz, J.; Ramírez-Velásquez, J.; Galindo-González, F. J.; Zavala-Castillo, J. C.; De León-Nava, M. A.; Robles-Díaz, G.; Escobedo, G.

    2013-01-01

    Interleukin-(IL-) 12 has been recently suggested to participate during development of insulin resistance in obese mice. Nevertheless, serum IL-12 levels have not been accurately determined in overweight and obese humans. We thus studied serum concentrations of IL-12 in Mexican adult individuals, examining their relationship with low-grade inflammation and obesity-related parameters. A total of 147 healthy individuals, 43 normal weight, 61 overweight, and 43 obese subjects participated in the study. Circulating levels of IL-12, tumor necrosis factor-alpha (TNF-α), leptin, insulin, glucose, total cholesterol, and triglyceride were measured after overnight fasting in all of the study subjects. Waist circumference and body fat percentage were recorded for all the participants. Serum IL-12 was significantly higher in overweight and obese individuals than in normal weight controls. Besides being strongly related with body mass index (r = 0.5154), serum IL-12 exhibited a significant relationship with abdominal obesity (r = 0.4481), body fat percentage (r = 0.5625), serum glucose (r = 0.3158), triglyceride (r = 0.3714), and TNF-α (r = 0.4717). Thus, serum levels of IL-12 are increased in overweight and obese individuals and show a strong relationship with markers of low-grade inflammation and obesity in the Mexican adult population. Further research is needed to understand the role of IL-12 in developing obesity-associated alterations in humans. PMID:23533314

  17. Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production.

    PubMed

    Vacaflores, Aldo; Freedman, Samantha N; Chapman, Nicole M; Houtman, Jon C D

    2017-01-01

    During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.

  18. IL-12 and IL-23 modulate plasticity of FoxP3(+) regulatory T cells in human Leprosy.

    PubMed

    Tarique, Mohd; Saini, Chaman; Naqvi, Raza Ali; Khanna, Neena; Sharma, Alpana; Rao, D N

    2017-03-01

    Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host's immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-β cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48h. Expression of FoxP3 in CD4(+)CD25(+) Tregs, intracellular cytokines IFN-γ, TGF-β, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3(+) and IL-17A(+) cells increase. rIL-12 and r-IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-β production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Sulforaphane protects from T cell-mediated autoimmune disease by inhibition of IL-23 and IL-12 in dendritic cells.

    PubMed

    Geisel, Julia; Brück, Jürgen; Glocova, Ivana; Dengler, Katja; Sinnberg, Tobias; Rothfuss, Oliver; Walter, Michael; Schulze-Osthoff, Klaus; Röcken, Martin; Ghoreschi, Kamran

    2014-04-15

    Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with anti-inflammatory properties. The published reports are best conceivable with an inhibition of T cell function, but the mode of action remains unknown. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis. Disease amelioration was associated with reduced IL-17 and IFN-γ expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DCs) inhibited TLR4-induced IL-12 and IL-23 production, and severely suppressed Th1 and Th17 development of T cells primed by SFN-treated DCs. SFN regulated the activity of the TLR4-induced transcription factor NF-κB, without affecting the degradation of its inhibitor IκB-α. Instead, SFN treatment of DCs resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with and thereby inhibits NF-κB p65. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 activity at the IL23a and IL12b promoters. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS. Thus, our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting NF-κB p65-regulated IL-23 and IL-12 expression.

  20. Cholera toxin subunit B inhibits IL-12 and IFN-{gamma} production and signaling in experimental colitis and Crohn's disease.

    PubMed

    Coccia, E M; Remoli, M E; Di Giacinto, C; Del Zotto, B; Giacomini, E; Monteleone, G; Boirivant, M

    2005-11-01

    Cholera toxin B subunit (CT-B) is a powerful modulator of immune responses. The authors have previously demonstrated that oral administration of recombinant CT-B (rCT-B) is able to prevent and cure the Crohn's disease (CD)-like trinitrobenzene sulfonic acid (TNBS) mediated colitis. In this study they extended their observations and examined if rCT-B interferes with the molecular signaling underlying the Th1 type response both in TNBS colitis and in ex vivo human CD explants. TNBS treated mice were fed with rCT-B, and IFN-gamma and IL-12 production by colonic lamina propria mononuclear cells (LPMC) was examined by ELISA. In vitro culture of mucosal explants from CD patients and non-inflammatory bowel disease controls, pre-incubated with rCT-B, were examined for IFN-gamma and IL-12 production by ELISA and semiquantitative reverse transcription polymerase chain reactions. STAT-1, -4, -6 activation and T-bet expression were examined following rCT-B treatment by western blotting both in TNBS treated mice and in human mucosal explants. rCT-B significantly reduced IL-12 and IFN-gamma secretion by LPMC from TNBS treated mice. Consistent with this, rCT-B inhibited both STAT-4 and STAT-1 activation and downregulated T-bet expression. Inhibition of Th1 signaling by CT-B associated with no change in IL-4 synthesis and expression of active STAT-6 indicating that rCT-B does not enhance Th2 cell responses. Moreover, in vitro treatment of CD mucosal explants with rCT-B resulted in reduced secretion of IL-12/IFN-gamma and inhibition of STAT-4/STAT-1 activation and T-bet expression. These studies indicate that CT-B inhibits mucosal Th1 cell signaling and suggest that rCT-B may be a promising candidate for CD therapy.

  1. TLR ligands of Lactobacillus sakei LK-117 isolated from seed mash for brewing sake are potent inducers of IL-12.

    PubMed

    Masuda, Yasuyuki; Takahashi, Toshinari; Yoshida, Kazutoshi; Nishitani, Yosuke; Mizuno, Masashi; Mizoguchi, Haruhiko

    2011-10-01

    Many studies have investigated the immunostimulatory effects of bacteria, such as the anti-allergic effects of lactic acid bacteria (LAB) and LAB-fermented milk. Importantly, these anti-allergic effects have been observed for both viable and nonviable bacteria. However, there are no reported immunological effects of LAB isolated from kimoto, the traditional yeast starter culture used for brewing sake, which also involves spontaneous lactate fermentation. In this study, we determined whether the Leuconostoc mesenteroides and Lactobacillus sakei bacterial strains obtained from kimoto affected the production of interleukin-12 (IL-12), an inducer of the T-helper type-1 immune response. By incubating autoclaved bacteria with J774.1 macrophage-like cells, we found that L. sakei LK-117 induced a sustained increase in IL-12p40 production. The IL-12-inducing ability of LK-117 was unaffected by anti-TLR2 neutralization and was entirely inhibited when the LK-117 cells were treated with RNase. When LK-117 cells were treated with M-1, an N-acetylmuramidase, at varying concentrations and for different periods of time, the ability of the bacteria to induce IL-12 decreased quickly. Although an active fraction could be prepared by chromatography from the soluble products of enzymolysis, the fraction's induction ability was <2% of that of intact organisms, and induction ability disappeared completely upon anti-TLR2 neutralization after treating the active fraction with RNase. These results suggest that single-stranded RNA released from cells that were disrupted by autoclaving might act as a TLR ligand and provide a novel mechanism in which heat-killed LAB could be used to regulate immune activity.

  2. Human Pegivirus (HPgV; formerly known as GBV-C) inhibits IL-12 dependent natural killer cell function.

    PubMed

    Chivero, Ernest T; Bhattarai, Nirjal; McLinden, James H; Xiang, Jinhua; Stapleton, Jack T

    2015-11-01

    Human Pegivirus (HPgV, formally GB virus C) infects lymphocytes and NK cells in vivo, and infection is associated with reduced T cell and NK cell activation in HIV-infected individuals. The mechanism by which HPgV inhibits NK cell activation has not been assessed. Following IL-12 stimulation, IFNγ expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects (p=0.02). In addition, HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited NK cell IL-12-mediated IFNγ release. E2 protein also inhibited Tyk2 activation following IL-12 stimulation. In contrast, cytolytic NK cell function was not altered by HPgV. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV persistence and reduced immune activation during HIV-coinfection. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.

  3. Toll-like receptors, IFN-γ and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load.

    PubMed

    Farias, María Victoria Nieto; Lendez, Pamela Anahí; Marin, Maia; Quintana, Silvina; Martínez-Cuesta, Lucía; Ceriani, María Carolina; Dolcini, Guillermina Laura

    2016-08-01

    Bovine leukemia virus (BLV) infection is widespread mainly in dairy cattle and 5-10% of infected animals will die due to lymphosarcoma; most cattle remain asymptomatic but 30% develop persistent lymphocytosis (PL). BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms which govern the control of viral dissemination will be desirable for the design of effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. We aimed to characterize cytokines and toll-like receptors (TLR) expression related to the proviral load profiles. IFN-γ and IL-12 mRNA expression level was significantly higher in PBMC from infected cattle (LPL n=6 and HPL n=7) compared to uninfected animals (n=5). While no significant differences were observed in IL-12 expression between LPL and HPL group, IFN-γ expression was significantly higher in LPL animals. Infected cattle exhibited higher expression levels of TLR3, 7-9. Animals with HPL had significantly higher expression of TLR7/8 than uninfected cattle. TLR8 and TLR9 were up-regulated in HPL group, and TLR3 was up-regulated in LPL group. This is the first report related to TLR gene expression in BLV infected cattle and represents evidence of the involvement of these receptors in BLV recognition. Further studies on different subpopulations of immune cells may help clarify their role in response to BLV and its consequences on viral dissemination. Copyright © 2016. Published by Elsevier Ltd.

  4. Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

    PubMed

    Hennemann, B; Tam, Y K; Tonn, T; Klingemann, H G

    1999-07-01

    Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

  5. [Purulent pericarditis and colonic infiltrating to Salmonella enteritidis complicated by acute intussusception in a case of IL-12Rβ1 deficiency].

    PubMed

    Ailal, F; Tazi, A; Bustamante, J; Picard, C; Najib, J; Casanova, J-L; Bousfiha, A A

    2014-12-01

    IL-12 receptor β1 deficiency (IL-12Rβ1) predisposes patients to mycobacteria and Salmonella infections. We report a case of IL-12Rβ1 deficiency with a fatal multi-resistant Salmonella enteritidis infection. This boy was born after from a consanguineous marriage, and diagnosed as having a IL-12Rβ1 deficiency since the age of 3 months. He presented with recurrent Salmonella enteritidis essentially digestive localization, complicated by purulent pericarditis at the same germ at the age of two and a half years. At the age of 3, a colonic infiltration due to a Salmonella enteritidis resistant to antibiotics, was complicated by acute intussusception, and the child died. The IL-12Rβ1 deficiency is considered as having a good prognosis, in contrast to what happened in our patient. We review therapeutic issues in these patients.

  6. Design of a Phase I Clinical Trial to Evaluate M032, a Genetically Engineered HSV-1 Expressing IL-12, in Patients with Recurrent/Progressive Glioblastoma Multiforme, Anaplastic Astrocytoma, or Gliosarcoma.

    PubMed

    Patel, Daxa M; Foreman, Paul M; Nabors, L Burt; Riley, Kristen O; Gillespie, G Yancey; Markert, James M

    2016-06-01

    M032 is a second-generation oncolytic herpes simplex virus (oHSV) that selectively replicates in tumor cells. M032 kills tumor cells directly through oncolytic replication and then proceeds to infect tumor cells in proximity, continuing the process of tumor destruction. In addition to this direct oncolytic activity, the virus carries a therapeutic payload-thus acting as a gene therapy vector-and causes the tumor cell to synthesize and secrete the immunity-stimulating protein interleukin-12 (IL-12) before cell death. (1) Human IL-12 is expressed and promotes an immune response against surviving tumor cells, increasing the antitumor effect of the therapy. IL-12 also produces an antiangiogenic effect, by interfering with the production of new tumor blood vessels necessary for tumor growth. Thus, M032 oHSV exerts antitumor effects through three distinct potential mechanisms. The virus has also been genetically engineered to minimize toxic effects for the patient. Preclinical animal models support the safety of intracranial inoculation with M032 in two relevant species (mouse and nonhuman primate). This clinical protocol outlines the dose-escalating phase I study for evaluation of M032 in patients with recurrent or progressive malignant glioma.

  7. Host Susceptibility to Brucella abortus Infection Is More Pronounced in IFN-γ knockout than IL-12/β2-Microglobulin Double-Deficient Mice

    PubMed Central

    Brandão, Ana Paula M. S.; Oliveira, Fernanda S.; Carvalho, Natalia B.; Vieira, Leda Q.; Azevedo, Vasco; Macedo, Gilson C.; Oliveira, Sergio C.

    2012-01-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. IFN-γ, IL-12, and CD8+ T lymphocytes are important components of host immune responses against B. abortus. Herein, IFN-γ and IL-12/β2-microglobulin (β2-m) knockout mice were used to determine whether CD8+ T cells and IL-12-dependent IFN-γ deficiency would be more critical to control B. abortus infection compared to the lack of endogenous IFN-γ. At 1 week after infection, IFN-γ KO and IL-12/β2-m KO mice showed increased numbers of bacterial load in spleens; however, at 3 weeks postinfection (p.i.), only IFN-γ KO succumbed to Brucella. All IFN-γ KO had died at 16 days p.i. whereas death within the IL-12/β2-m KO group was delayed and occurred at 32 days until 47 days postinfection. Susceptibility of IL-12/β2-m KO animals to Brucella was associated to undetectable levels of IFN-γ in mouse splenocytes and inability of these cells to lyse Brucella-infected macrophages. However, the lack of endogenous IFN-γ was found to be more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies. PMID:22194770

  8. Profiles of IFN-γ and its regulatory cytokines (IL-12, IL-18 and IL-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis

    PubMed Central

    LEE, J-S; SONG, C-H; KIM, C-H; KONG, S-J; SHON, M-H; KIM, H-J; PARK, J-K; PAIK, T-H; JO, E-K

    2002-01-01

    This study investigated the profiles of IFN-γ and its regulatory cytokines (IL-12, IL-18 and IL-10) in response to a purified protein derivative (PPD) antigen in peripheral blood mononuclear cells (PBMC) from 18 HIV-negative patients with multidrug-resistant tuberculosis (MDRTB), and compared them with those from 19 healthy tuberculin reactors (HTR). ELISA results showed that following stimulation with PPD, IFN-γ production was significantly reduced, whereas production of both IL-18 and IL-10 was significantly elevated in MDRTB patients compared with HTR. Three out of 18 patients with MDRTB of greater than 4 years duration showed significantly elevated IL-12 p70 production, induced by in vitro PPD stimulation of their PBMC, when compared with data from HTR. However, when taken as a group, MDRTB patients were similar to HTR in their IL-12 p70-producing capacity. IL-12 p70 protein paralleled IL-12 p40 protein expression. In addition, the production of IL-12 p40 was significantly correlated with IL-10 in all patients, but was not correlated with IFN-γ. Neutralization of IL-10 increased IL-12 p40 about twofold, but did not significantly alter IFN-γ induction in MDRTB. IFN-γ in MDRTB was highly correlated with lymphoproliferation and CD4 counts, but was not correlated with IL-12, IL-18 or IL-10 production. Our findings suggest that patients with MDRTB have dysregulated IL-12, IL-18 and IL-10 production during Mycobacterium tuberculosis infection, and the cytokine profiles are similar to those in patients with drug-sensitive advanced TB previously reported in the literature. In addition, IL-10 may not have a dominant role in defective IFN-γ production in patients with MDRTB. PMID:12067307

  9. IL-12 promotes myeloid-derived suppressor cell recruitment and bacterial persistence during Staphylococcus aureus orthopedic implant infection.

    PubMed

    Heim, Cortney E; Vidlak, Debbie; Scherr, Tyler D; Hartman, Curtis W; Garvin, Kevin L; Kielian, Tammy

    2015-04-15

    Staphylococcus aureus is a leading cause of human prosthetic joint infections (PJIs) typified by biofilm formation. We recently identified a critical role for myeloid-derived suppressor cells (MDSCs) in S. aureus biofilm persistence. Proinflammatory signals induce MDSC recruitment and activation in tumor models; however, the mechanisms responsible for MDSC homing to sites of biofilm infection are unknown. In this study, we report that several cytokines (IL-12p40, IL-1β, TNF-α, and G-CSF) and chemokines (CXCL2, CCL5) were significantly elevated in a mouse model of S. aureus PJI. This coincided with significantly increased MDSC infiltrates concomitant with reduced monocyte, macrophage, and T cell influx compared with uninfected animals. Of the cytokines detected, IL-12 was of particular interest based on its ability to possess either pro- or anti-inflammatory effects mediated through p35-p40 heterodimers or p40 homodimers, respectively. MDSC recruitment was significantly reduced in both p40 and p35 knockout mice, which resulted in enhanced monocyte and neutrophil influx and bacterial clearance. Adoptive transfer of wild-type MDSCs into infected p40 knockout animals worsened disease outcome, as evidenced by the return of S. aureus burdens to levels typical of wild-type mice. Tissues obtained from patients undergoing revision surgery for PJI revealed similar patterns of immune cell influx, with increased MDSC-like cells and significantly fewer T cells compared with aseptic revisions. These findings reveal a critical role for IL-12 in shaping the anti-inflammatory biofilm milieu by promoting MDSC recruitment.

  10. Cerebrospinal Fluid IL-12p40, CXCL13 and IL-8 as a Combinatorial Biomarker of Active Intrathecal Inflammation

    PubMed Central

    Bielekova, Bibiana; Komori, Mika; Xu, Quangang; Reich, Daniel S.; Wu, Tianxia

    2012-01-01

    Diagnosis and management of the neuroinflammatory diseases of the central nervous system (CNS) are hindered by the lack of reliable biomarkers of active intrathecal inflammation. We hypothesized that measuring several putative inflammatory biomarkers simultaneously will augment specificity and sensitivity of the biomarker to the clinically useful range. Based on our pilot experiment in which we measured 18 inflammatory biomarkers in 10-fold concentrated cerebrospinal fluid (CSF) derived from 16 untreated patients with highly active multiple sclerosis (MS) we selected a combination of three CSF biomarkers, IL-12p40, CXCL13 and IL-8, for further validation. Concentrations of IL-12p40, CXCL13 and IL-8 were determined in a blinded fashion in CSF samples from an initial cohort (n = 72) and a confirmatory cohort (n = 167) of prospectively collected, untreated subjects presenting for a diagnostic work-up of possible neuroimmunological disorder. Diagnostic conclusion was based on a thorough clinical workup, which included laboratory assessment of the blood and CSF, neuroimaging and longitudinal follow-up. Receiver operating characteristic (ROC) curve analysis in conjunction with principal component analysis (PCA), which was used to combine information from all three biomarkers, assessed the diagnostic value of measured biomarkers. Each of the three biomarkers was significantly increased in MS and other inflammatory neurological disease (OIND) in comparison to non-inflammatory neurological disorder patients (NIND) at least in one cohort. However, considering all three biomarkers together improved accuracy of predicting the presence of intrathecal inflammation to the consistently good to excellent range (area under the ROC curve = 0.868–0.924). Future clinical studies will determine if a combinatorial biomarker consisting of CSF IL-12p40, CXCL13 and IL-8 provides utility in determining the presence of active intrathecal inflammation in diagnostically uncertain

  11. [Immunomodulation of Uncaria tomentosa over dendritic cells, il-12 and profile TH1/TH2/TH17 in breast cancer].

    PubMed

    Núñez, César; Lozada-Requena, Iván; Ysmodes, Tíndara; Zegarra, Daniel; Saldaña, Fatima; Aguilar, José

    2015-10-01

    Objetives. This study aimed to research the in vitro immunomodulatory effects of an Uncaria tomentosa hydroalcoholic extract standardized (5.03%, pentacyclic oxindole alkaloids) (UT-POA) on the immunophenotype of dendritic cells (DC) subsets, Th1, Th2, Th17 and IL-12 cytokines from patients with stage II breast cancer (BCII) and healthy women (H). Blood of 11 H and 7 BCII was obtained, PBMC were isolated and cultured for 2h with/without various concentrations of UT-POA and stimulated or not with LPS for 24h. PBMC were labeled with specific antibodies for DC and in the supernatant we measured Th1/Th2/Th17 cytokines, both by flow cytometry. Furthermore IL-12 was measured by ELISA. UT-POA did not alter DC or accessory molecules expression in BCII. However, H exhibited a decrease in the percentage of mDC (myeloid DC) and an increase in HLA-DR and CD86 expression at 1000 μg/mL. IL-12 secretion was modified only in the H group, increasing p70 subunit and decreasing p40 subunit. UT-POA increased Th1 (IFN-γ and IL-2), Th2 (IL-4) and Th17 (IL-17) secretion in both groups. UT-POA increased the production of cytokines related with anti-tumoral response at concentrations of 500-1000 μg/mL. This positive effect should be evaluated not only systemically but also in the tumor microenvironment in further studies. UT-POA may be a useful phytochemical in chemoprevention and in the alternative use in cancer therapies.

  12. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    PubMed Central

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  13. Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27.

    PubMed

    Zwirner, Norberto Walter; Ziblat, Andrea

    2017-01-01

    Natural killer (NK) cells are characterized by their ability to detect and induce apoptosis of susceptible target cells and by secretion of immunoregulatory cytokines such as IFN-γ. Activation of these effector functions is triggered upon recognition of tumor and pathogen (mostly virus)-infected cells and because of a bidirectional cross talk that NK cells establish with other cells of myeloid origin such as dendritic cells (DC) and macrophages. A common characteristic of these myeloid cells is their ability to secrete different members of the IL-12 family of cytokines such as IL-12, IL-23, and IL-27 and cytokines such as IL-15 and IL-18. Although the effect of IL-12, IL-15, and IL-18 has been characterized, the effect of IL-23 and IL-27 on NK cells (especially human) remains ill-defined. Particularly, IL-27 is a cytokine with dual functions as it has been described as pro- and as anti-inflammatory in different experimental settings. Recent evidence indicates that this cytokine indeed promotes human NK cell activation, IFN-γ secretion, NKp46-dependent NK cell-mediated cytotoxicity, and antibody (Ab)-dependent NK cell-mediated cytotoxicity (ADCC) against monoclonal Ab-coated tumor cells. Remarkably, IL-27 also primes NK cells for IL-18 responsiveness, enhancing these functional responses. Consequently, IL-27 acts as a pro-inflammatory cytokine that, in concert with other DC-derived cytokines, hierarchically contributes to NK cells activation and effector functions, which likely contributes to foster the adaptive immune response in different physiopathological conditions.

  14. Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27

    PubMed Central

    Zwirner, Norberto Walter; Ziblat, Andrea

    2017-01-01

    Natural killer (NK) cells are characterized by their ability to detect and induce apoptosis of susceptible target cells and by secretion of immunoregulatory cytokines such as IFN-γ. Activation of these effector functions is triggered upon recognition of tumor and pathogen (mostly virus)-infected cells and because of a bidirectional cross talk that NK cells establish with other cells of myeloid origin such as dendritic cells (DC) and macrophages. A common characteristic of these myeloid cells is their ability to secrete different members of the IL-12 family of cytokines such as IL-12, IL-23, and IL-27 and cytokines such as IL-15 and IL-18. Although the effect of IL-12, IL-15, and IL-18 has been characterized, the effect of IL-23 and IL-27 on NK cells (especially human) remains ill-defined. Particularly, IL-27 is a cytokine with dual functions as it has been described as pro- and as anti-inflammatory in different experimental settings. Recent evidence indicates that this cytokine indeed promotes human NK cell activation, IFN-γ secretion, NKp46-dependent NK cell-mediated cytotoxicity, and antibody (Ab)-dependent NK cell-mediated cytotoxicity (ADCC) against monoclonal Ab-coated tumor cells. Remarkably, IL-27 also primes NK cells for IL-18 responsiveness, enhancing these functional responses. Consequently, IL-27 acts as a pro-inflammatory cytokine that, in concert with other DC-derived cytokines, hierarchically contributes to NK cells activation and effector functions, which likely contributes to foster the adaptive immune response in different physiopathological conditions. PMID:28154569

  15. TNFR2 maintains adequate IL-12 production by dendritic cells in inflammatory responses by regulating endogenous TNF levels.

    PubMed

    Martin, Elisabeth M; Remke, Annika; Pfeifer, Eva; Polz, Johannes; Pietryga-Krieger, Anne; Steffens-Weber, Dorothea; Freudenberg, Marina A; Mostböck, Sven; Männel, Daniela N

    2014-10-01

    Sepsis-induced immune reactions are reduced in TNF receptor 2 (TNFR2)-deficient mice as previously shown. In order to elucidate the underlying mechanisms, the functional integrity of myeloid cells of TNFR2-deficient mice was analyzed and compared to wild type (WT) mice. The capacity of dendritic cells to produce IL-12 was strongly impaired in TNF-deficient mice, mirroring impaired production of IL-12 by WT dendritic cells in sepsis or after LPS or TNF pre-treatment. In addition, TNFR2-deficient mice were refractory to LPS pre-treatment and also to hyper-sensitization by inactivated Propionibacterium acnes, indicating habituation to inflammatory stimuli by the immune response when TNFR2 is lacking. Constitutive expression of TNF mRNA in kidney, liver, spleen, colon and lung tissue, and the presence of soluble TNFR2 in urine of healthy WT mice supported the conclusion that TNF is continuously present in naïve mice and controlled by soluble TNFR2. In TNFR2-deficient mice endogenous TNF levels cannot be balanced and the continuous exposure to enhanced TNF levels impairs dendritic cell function. In conclusion, TNF pre-exposure suppresses secondary inflammatory reactions of myeloid cells; therefore, continuous control of endogenous TNF by soluble TNFR2 seems to be essential for the maintenance of adequate sensitivity to inflammatory stimuli.

  16. IL-12-polarized Th1 cells produce GM-CSF and induce EAE independent of IL-23

    PubMed Central

    Grifka-Walk, Heather M.; Giles, David A.; Segal, Benjamin M.

    2016-01-01

    CD4+ T-helper (Th) cells reactive against myelin antigens mediate the animal model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL-23 is universally required for the acquisition of pathogenic properties by myelin-reactive T cells. We show that IL-12-modulated Th1 cells readily produce IFN-γ and GM-CSF in the central nervous system (CNS) and induce a severe form of EAE via an IL-23-independent pathway. Th1-mediated EAE is characterized by monocyte-rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2-dependent. Conversely, IL-23-modulated, stable Th17 cells induce EAE with a relatively mild course via an IL-12-independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4+ T helper cell subsets and polarizing factors. PMID:26220255

  17. Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines

    PubMed Central

    Simova, Jana; Sapega, Olena; Imrichova, Terezie; Stepanek, Ivan; Kyjacova, Lenka; Mikyskova, Romana; Indrova, Marie; Bieblova, Jana; Bubenik, Jan; Bartek, Jiri; Hodny, Zdenek; Reinis, Milan

    2016-01-01

    Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo. PMID:27448982

  18. Evaluation of IL-12 serum level in patients with recalcitrant multiple common warts, treated by intralesional tuberculin antigen.

    PubMed

    Abd-Elazeim, Fian M A; Mohammed, Ghada F A; Fathy, Amal; Mohamed, Roshdy W

    2014-06-01

    No universal consensus about optimal modality for treating the recalcitrant multiple common warts (RMCW). To evaluate the immunological mechanisms and clinical therapeutic effect of using of intralesional purified protein derivative (PPD) in the treatment of RMCW. The study included 40 patients with RMCW. They were randomly assigned to 2 groups: first group (20 patients) received intralesional PPD antigen, and second group (20 patients) received intralesional saline as a control group. In both groups, injections were made into single lesions, or largest wart in case of multiple lesions, at weekly intervals, until complete clearance or for a maximum of six treatments. Blood serum was taken at pre-study and at week 6 to measure IL-12 level. A significant difference was found between the therapeutic responses of RMCW to PPD antigen and saline control group (p < 0.001). In the PPD group, complete response was achieved in 75% after 5.8 ± 0.7 sessions' patients presenting with RMCW. There was a statistically significant increase in IL-12 of PPD group versus saline group. No recurrence was observed in the PPD group. Intralesional immunotherapy by PPD antigen is an effective and a safe treatment for RMCW in previously immunized patients.

  19. Dietary grape-seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12.

    PubMed

    Sharma, Som D; Katiyar, Santosh K

    2006-01-01

    We have shown previously that dietary grape seed proanthocyanidins (GSPs) inhibit UVB-induced photocarcinogenesis in mice. As UVB-induced immune suppression has been implicated in the development of skin cancer risk, we investigated whether dietary GSPs can modulate the effects of UVB on the immune system. We found that the UVB-induced (180 mJ/cm2) ear swelling response (inflammatory reaction) was significantly lower in mice fed with a GSP-supplemented (0.5 and 1.0%, w/w) diet than mice fed with the standard AIN76A diet. Dietary GSPs markedly inhibited UVB-induced (180 mJ/cm2) suppression of contact hypersensitivity responses in a local model of immunosuppression but had only moderate inhibitory effect in a systemic model of immunosuppression. Dietary GSPs reduced the UVB-induced increase in immunosuppressive cytokine interleukin (IL)-10 in skin and draining lymph nodes compared with mice that did not receive GSPs. In contrast, GSPs enhanced the production of immunostimulatory cytokine IL-12 in the draining lymph nodes. Intraperitoneal injection of GSPs-fed mice with a neutralizing anti-IL-12 antibody abrogated the protective effects of the GSPs against UVB-induced suppression of the contact hypersensitivity response. These data indicate for the first time that GSPs modulate UVB-induced immunosuppression and suggest that this may be one of the possible mechanisms by which they prevent photocarcinogenesis in mice.

  20. Photodynamic therapy with recombinant adenovirus AdmIL-12 enhances anti-tumour therapy efficacy in human papillomavirus 16 (E6/E7) infected tumour model

    PubMed Central

    Park, Eun Kyung; Bae, Su-Mi; Kwak, Sun-Young; Lee, Sung Jong; Kim, Yong-Wook; Han, Chan-Hee; Cho, Hyun-Jung; Kim, Kyung Tae; Kim, Young-Jae; Kim, Hyun-Jung; Ahn, Woong Shick

    2008-01-01

    Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. Here we investigated the utility of adenoviral delivery of interleukin-12 (AdmIL-12) as an adjuvant for PDT in mouse tumour challenge model. PDT was performed by irradiating Radachlorin in C57BL/6 mice transplanted with TC-1 cells. PDT plus AdmIL-12 treatment for tumour suppression as well as specific immune responses were evaluated with the following tests: in vitro and in vivo tumour growth inhibition, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) assay, and cytotoxic T lymphocyte (CTL) assay. Direct intratumoral injection of AdmIL-12 resulted in a significant suppression of tumour growth compared to the control group. Treatment of PDT along with AdmIL-12 further enhanced antitumour effects significantly higher than either AdmIL-12 or PDT alone. This combined treatment resulted in complete regression of 9-mm sized tumour in every animal. We also evaluated immune responses induced by these treatments. Combined treatment significantly increased the production level of IFN-γ and TNF-α compared with that by AdmIL-12 or PDT alone. PDT plus AdmIL-12 enhanced antitumour immunity through increased expansion of the CTL subset mediated by CD8+ T cells. Taken together, these results indicate that the high anti-cancer activity of PDT with AdmIL-12 is a powerful tool against cancer therapy and is a promising subject for further investigation. PMID:18397271

  1. Ginger extract modulates the expression of IL-12 and TGF-β in the central nervous system and serum of mice with experimental autoimmune encephalomyelitis

    PubMed Central

    Jafarzadeh, Abdollah; Ahangar-Parvin, Reyhane; Nemat, Maryam; Taghipour, Zahra; Shamsizadeh, Ali; Ayoobi, Fatemeh; Hassan, Zuhair Mohammad

    2017-01-01

    Objective: The main function of IL-12 is differentiation of naive T cells intoTh1 cells and TGF-β is a powerful immunoregulatory cytokine. The immunomodulatory and anti-inflammatory properties of ginger have also been reported in some studies. The aim of this study was to evaluate the effects of ginger extract on the expression of IL-12 and TGF-β in a model of experimental autoimmune encephalomyelitis (EAE). Materials and Methods: EAE was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein emulsified in complete Freund's adjuvant. The mice were administered intra-peritoneally with ginger extracts or PBS, from day +3 to +30. On day 31, mice were scarified and the expression of IL-12 and TGF-β mRNA in the spinal cord were determined by using real time-PCR. The serum levels of cytokines were measured by ELISA. Results: In PBS-treated EAE mice, the expression of IL-12 P35 and IL-12 P40 mRNA in the CNS and the mean serum levels of IL-12 were significantly higher than those of healthy group (p<0.001). In ginger-treated EAE mice, the expression of IL-12 mRNA and its serum levels were significantly lower as compared to PBS-treated EAE mice. No significant difference was observed between PBS-treated EAE mice and healthy group regarding the expression of TGF-β mRNA. In ginger (300 mg/kg)-treated EAE group, the expression of TGF-β mRNA and its serum levels were significantly higher in comparison to PBS-treated EAE mice (p<0.01 and p<0.05, respectively). Conclusion: These results indicated that ginger extract modulates the expression of IL-12 and TGF-β in CNS and serum of EAE mice. PMID:28265547

  2. Vaccination with a preparation based on recombinant cysteine peptidases and canine IL-12 does not protect dogs from infection with Leishmania infantum.

    PubMed

    Poot, J; Spreeuwenberg, K; Sanderson, S J; Schijns, V E C J; Mottram, J C; Coombs, G H; Vermeulen, A N

    2006-03-24

    Cysteine peptidases (CPs) have been implicated in various processes central to the pathogenicity of Leishmania parasites, and are thought to be key factors in the host-parasite interaction. In order to fully evaluate the potential of the CPs as vaccine candidates, studies in natural host species are required. In the study we report here, recombinant L. infantum CPs CPA and CPB were used to vaccinate dogs. In order to induce an appropriate response against the antigens, recombinant canine IL-12 was added as an adjuvant either by itself or in combination with Quil A. After vaccination, dogs were given an intravenous challenge with promastigotes of L. infantum JPC strain. In both vaccinated groups (CPs with IL-12 or CPs with IL-12 and Quil A) CP-specific antibodies were detected after vaccination, indicating that there was a reaction to the vaccine. However, all dogs were found parasite-positive and all developed some degree of clinical leishmaniosis. The observed lack of efficacy of the candidate vaccines could be due, completely or in part, to a number of factors associated with the vaccine antigen, the adjuvant or host-parasite interactions. When compared to results from other studies, it seems less likely that the molecular conformation of the rCPs or rIL-12 caused this lack of efficacy. More plausible explanations are the dose and timing of the IL-12 application and the potentially different effects IL-12 induces as an adjuvant in either the murine or the canine leishmaniosis model.

  3. The increasingly anti-tumor effect of a colonic carcinoma DNA vaccine carrying HER2 by the adjuvanticity of IL-12.

    PubMed

    Gao, Ping; Zhang, Chunhua; Bian, Xiaoxia; Guo, Yanjun; Wei, Yueguang; Zhang, Li; Liu, Zhaoyang; Wang, Xiuying; Huang, Shumin

    2016-09-23

    The present study aimed to determine the effect of recombinant DNA vaccine-based human epidermal growth factor receptor-2 (HER2) and Interleukin 12 (IL-12) on the development of colonic carcinoma in mice and the potential immune mechanisms involved. Recombinant plasmids pVAX1-HER2, pVAX1-IL-12 and pVAX1-HER2-IL-12 were constructed, and injected into female mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were examined by enzyme linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively. To evaluate the anti-tumor efficacy of the plasmids, a mouse model with a HER2-expressing tumor was designed. Mice vaccinated with the HER2-IL-12 plasmid generated the strongest inhibition efficacy on the growth of HER2-expressing tumors and prolonged mouse survival. These observations emphasized the potential of IL-12 as an adjuvant for DNA vaccines and of vaccines based on HER2 and IL-12 as a promising treatment for colonic carcinoma.

  4. Genetically determined high activity of IL-12 and IL-18 in ulcerative colitis and TLR5 in Crohns disease were associated with non-response to anti-TNF therapy.

    PubMed

    Bank, S; Andersen, P S; Burisch, J; Pedersen, N; Roug, S; Galsgaard, J; Turino, S Y; Brodersen, J B; Rashid, S; Rasmussen, B K; Avlund, S; Olesen, T B; Hoffmann, H J; Nexø, B A; Sode, J; Vogel, U; Andersen, V

    2017-01-31

    Anti-tumour necrosis factor-α (TNF-α) is used for treatment of severe cases of inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). However, one-third of the patients do not respond to the treatment. A recent study indicated that genetically determined high activity of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6 and interferon gamma (IFN-γ), are associated with non-response to anti-TNF therapy. Using a candidate gene approach, 21 functional single-nucleotide polymorphisms (SNPs) in 14 genes in the Toll-like receptors, the inflammasome and the IFNG pathways were assessed in 482 and 256 prior anti-TNF naïve Danish patients with CD and UC, respectively. The results were analysed using logistic regression (adjusted for age and gender). Eight functional SNPs were associated with anti-TNF response either among patients with CD (TLR5 (rs5744174) and IFNGR2 (rs8126756)), UC (IL12B (rs3212217), IL18 (rs1946518), IFNGR1 (rs2234711), TBX21 (rs17250932) and JAK2 (rs12343867)) or in the combined cohort of patient with CD and UC (IBD) (NLRP3 (rs10754558), IL12B (rs3212217) and IFNGR1 (rs2234711)) (P<0.05). Only the association with heterozygous genotype of IL12B (rs3212217) (OR: 0.24, 95% CI: 0.11-0.53, P=0.008) among patients with UC withstood Bonferroni correction for multiple testing. In conclusion, Our results suggest that SNPs associated with genetically determined high activity of TLR5 among patients with CD and genetically determined high IL-12 and IL-18 levels among patients with UC were associated with non-response. Further studies will evaluate whether these genes may help stratifying patients according to the expected response to anti-TNF treatment.The Pharmacogenomics Journal advance online publication, 31 January 2017; doi:10.1038/tpj.2016.84.

  5. The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice.

    PubMed

    Cheng, Yu-Li; Song, Wen-Jian; Liu, Wen-Qi; Lei, Jia-Hui; Kong, Zheng; Li, Yong-Long

    2012-01-01

    CD4(+) T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 μm vs. 294.4 ± 72.2 μm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.

  6. Molecules altering the intracellular thiol content modulate NF-kB and STAT-1/IRF-1 signalling pathways and IL-12 p40 and IL-27 p28 production in murine macrophages.

    PubMed

    Fraternale, Alessandra; Crinelli, Rita; Casabianca, Anna; Paoletti, Maria Filomena; Orlandi, Chiara; Carloni, Elisa; Smietana, Michaël; Palamara, Anna Teresa; Magnani, Mauro

    2013-01-01

    The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.

  7. Interferon-gamma (IFN-gamma)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans.

    PubMed

    Kawakami, K; Qureshi, M H; Zhang, T; Koguchi, Y; Shibuya, K; Naoe, S; Saito, A

    1999-07-01

    We have recently demonstrated that IL-12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN-gamma in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL-12 on survival of infected mice and prevented IL-12-induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL-12 was clearly inhibited by anti-IFN-gamma MoAb. We also examined the local production of mononuclear cell-attracting chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and IFN-gamma-inducible protein 10 (IP-10) in the lungs using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL-12 treatment markedly induced their production. Interestingly, neutralizing anti-IFN-gamma MoAb strongly suppressed IL-12-induced production of these chemokines. Similar results were obtained with MCP-1 and MIP-1alpha when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN-gamma plays a central role in the protective effects of IL-12 by inducing mononuclear leucocyte-attracting chemokines and cellular inflammatory responses.

  8. IL-12/15/18-preactivated NK cells suppress GvHD in a mouse model of mismatched hematopoietic cell transplantation.

    PubMed

    Hüber, Christian M; Doisne, Jean-Marc; Colucci, Francesco

    2015-06-01

    Mismatched hematopoietic cell transplants for treating leukemia are complicated by graft versus host disease (GvHD). Here, we show that adoptively transferred IL-12/15/18-preactivated NK cells suppress GvHD in a mouse model of fully mismatched hematopoietic cell transplantation. These IL-12/15/18-preactivated NK cells maintained Eomesodermin (Eomes) and T-bet expression upon transfer and, while there was no evidence of direct killing of donor T cells or host DCs by the IL-12/15/18-preactivated NK cells, proliferation of donor T cells was inhibited. Strikingly, the graft versus leukemia effect mediated by donor T cells was retained, resulting in improved overall survival of mice that received lymphoma cells, donor allogeneic T cells, and IL-12/15/18-preactivated NK cells. These results suggest that IL-12/15/18-preactivated NK cells may be useful in improving immunotherapy of mismatched hematopoietic cell transplantation. Compared with previously proposed protocols, our findings suggest that in vitro NK-cell preactivation with this cytokine cocktail offers the significant advantage that cytokines do not need to be administered systemically to sustain NK-cell activity, thus avoiding toxicity.

  9. IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

    PubMed

    Napione, Lucia; Strasly, Marina; Meda, Claudia; Mitola, Stefania; Alvaro, Maria; Doronzo, Gabriella; Marchiò, Serena; Giraudo, Enrico; Primo, Luca; Arese, Marco; Bussolino, Federico

    2012-12-01

    Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

  10. An Il12-Il2-Antibody Fusion Protein Targeting Hodgkin's Lymphoma Cells Potentiates Activation Of Nk And T Cells For An Anti-Tumor Attack

    PubMed Central

    Friedrichs, Björn; Heuser, Claudia; Guhlke, Stefan; Abken, Hinrich; Hombach, Andreas A.

    2012-01-01

    Successful immunotherapy of Hodgkin's disease is so far hampered by the striking unresponsiveness of lymphoma infiltrating immune cells. To mobilize both adoptive and innate immune cells for an anti-tumor attack we fused the pro-inflammatory cytokines IL2 and IL12 to an anti-CD30 scFv antibody in a dual cytokine fusion protein to accumulate both cytokines at the malignant CD30+ Hodgkin/Reed-Sternberg cells in the lymphoma lesion. The tumor-targeted IL12-IL2 fusion protein was superior in activating resting T cells to amplify and secrete pro-inflammatory cytokines compared to targeted IL2 or IL12 alone. NK cells were also activated by the dual cytokine protein to secrete IFN-γ and to lyse target cells. The tumor-targeted IL12-IL2, when applied by i.v. injection to immune-competent mice with established antigen-positive tumors, accumulated at the tumor site and induced tumor regression. Data demonstrate that simultaneous targeting of two cytokines in a spatial and temporal simultaneous fashion to pre-defined tissues is feasible by a dual-cytokine antibody fusion protein. In the case of IL12 and IL2, this produced superior anti-tumor efficacy implying the strategy to muster a broader immune cell response in the combat against cancer. PMID:23028547

  11. Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: Effect of IL-12, dose, and route

    PubMed Central

    Mealey, R.H.; Stone, D.M.; Hines, M.T.; Alperin, D.C.; Littke, M.H.; Leib, S.R.; Leach, S.E.; Hines, S.A.

    2012-01-01

    Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates. PMID:17889970

  12. Leishmania major inhibits IL-12 in macrophages by signaling through CR3 (CD11b/CD18) and downregulation of ETS-mediated transcription

    PubMed Central

    Ricardo-Carter, Cristina; Favila, Michelle; Polando, Rachel E.; Cotton, Rachel N.; Horner, Kimberly Bogard; Condon, David; Ballhorn, Wibke; Whitcomb, James P.; Yadav, Mahesh; Geister, Rebecca L.; Schorey, Jeffery S.; McDowell, Mary Ann

    2013-01-01

    SUMMARY Leishmania major is an etiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives anti-leishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared to WT cells upon LPS stimulation. We therefore investigated multiple signaling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1, or IRF-8 activation in WT or CD11b deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation, nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major mediated inhibition of IL-12 occurs through CR3 engagement, however the mechanism of inhibition is independent of NFκB, MAPK, IRF, and ETS. PMID:23834512

  13. Leishmania major inhibits IL-12 in macrophages by signalling through CR3 (CD11b/CD18) and down-regulation of ETS-mediated transcription.

    PubMed

    Ricardo-Carter, C; Favila, M; Polando, R E; Cotton, R N; Bogard Horner, K; Condon, D; Ballhorn, W; Whitcomb, J P; Yadav, M; Geister, R L; Schorey, J S; McDowell, M A

    2013-12-01

    Leishmania major is an aetiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives antileishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared with WT cells upon lipopolysaccharide (LPS) stimulation. We therefore investigated multiple signalling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1 or IRF-8 activation in WT or CD11b-deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major-mediated inhibition of IL-12 occurs through CR3 engagement; however, the mechanism of inhibition is independent of NFκB, MAPK, IRF and ETS. © 2013 John Wiley & Sons Ltd.

  14. The Bacterial Preparation OK432 Induces IL-12p70 Secretion in Human Dendritic Cells in a TLR3 Dependent Manner

    PubMed Central

    Hovden, Arnt-Ove; Karlsen, Marie; Jonsson, Roland; Appel, Silke

    2012-01-01

    Dendritic cells (DC) used in therapeutic cancer immunotherapy have to be able to stimulate T cells resulting in an immune response that can efficiently target the cancer cells. One of the critical hurdles has been the lack of IL-12p70 production when maturating the DC, which is rectified by using the bacterial preparation OK432 (trade name Picibanil) to mature the cells. In order to identify the mechanism behind OK432 stimulation of DC, we investigated the contribution of different TLR to examine their involvement in IL-12p70 production. By combining different inhibitors of TLR signaling, we demonstrate here that TLR3 is responsible for the IL-12p70 production of DC induced by OK432. Moreover, our data suggest that the ligand triggering IL-12p70 secretion upon TLR3 stimulation is sensitive to proteinase and partly also RNAse treatment. The fact that a bacterial compound like OK432 can activate the TLR3 pathway in human DC is a novel finding. OK432 demonstrates a critical ability to induce IL-12p70 production, which is of great relevance in DC based cancer immunotherapy. PMID:22363584

  15. The bacterial preparation OK432 induces IL-12p70 secretion in human dendritic cells in a TLR3 dependent manner.

    PubMed

    Hovden, Arnt-Ove; Karlsen, Marie; Jonsson, Roland; Appel, Silke

    2012-01-01

    Dendritic cells (DC) used in therapeutic cancer immunotherapy have to be able to stimulate T cells resulting in an immune response that can efficiently target the cancer cells. One of the critical hurdles has been the lack of IL-12p70 production when maturating the DC, which is rectified by using the bacterial preparation OK432 (trade name Picibanil) to mature the cells. In order to identify the mechanism behind OK432 stimulation of DC, we investigated the contribution of different TLR to examine their involvement in IL-12p70 production. By combining different inhibitors of TLR signaling, we demonstrate here that TLR3 is responsible for the IL-12p70 production of DC induced by OK432. Moreover, our data suggest that the ligand triggering IL-12p70 secretion upon TLR3 stimulation is sensitive to proteinase and partly also RNAse treatment. The fact that a bacterial compound like OK432 can activate the TLR3 pathway in human DC is a novel finding. OK432 demonstrates a critical ability to induce IL-12p70 production, which is of great relevance in DC based cancer immunotherapy.

  16. Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: effect of IL-12, dose, and route.

    PubMed

    Mealey, R H; Stone, D M; Hines, M T; Alperin, D C; Littke, M H; Leib, S R; Leach, S E; Hines, S A

    2007-10-23

    Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.

  17. Randomized comparison of single dose of recombinant human IL-12 versus placebo for restoration of hematopoiesis and improved survival in rhesus monkeys exposed to lethal radiation

    PubMed Central

    2014-01-01

    Background The hematopoietic syndrome of the acute radiation syndrome (HSARS) is a life-threatening condition in humans exposed to total body irradiation (TBI); no drugs are approved for treating this condition. Recombinant human interleukin-12 (rHuIL-12) is being developed for HSARS mitigation under the FDA Animal Rule, where efficacy is proven in an appropriate animal model and safety is demonstrated in humans. Methods In this blinded study, rhesus monkeys (9 animals/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-12 at doses of 50, 100, 250, or 500 ng/kg (groups 2–5, respectively), without antibiotics, fluids or blood transfusions, 24–25 hours after TBI (700 cGy). Results Survival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1–5, respectively (log rank p < 0.05 for each dose vs. control). rHuIL-12 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-12 than in controls. Conclusions Data from this study demonstrate that a single injection of rHuIL-12 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-12 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure. PMID:24708888

  18. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    PubMed

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.

  19. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    PubMed Central

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-01-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  20. Immunotherapeutic Synergy Between Anti-CD137 mAb and Intratumoral Administration of a Cytopathic Semliki Forest Virus Encoding IL-12

    PubMed Central

    Quetglas, José I; Dubrot, Juan; Bezunartea, Jaione; Sanmamed, Miguel F; Hervas-Stubbs, Sandra; Smerdou, Cristian; Melero, Ignacio

    2012-01-01

    Intratumoral injection of Semliki Forest virus encoding interleukin-12 (SFV-IL-12) combines acute expression of IL-12 and stressful apoptosis of infected malignant cells. Agonist antibodies directed to costimulatory receptor CD137 (4-1BB) strongly amplify pre-existing cellular immune responses toward weak tumor antigens. In this study, we provide evidence for powerful synergistic effects of a combined strategy consisting of intratumoral injection of SFV-IL-12 and systemic delivery of agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8β+ T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic in vivo enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating CD8+ T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress. PMID:22735380

  1. Enhanced acute immune response in IL-12p35-/- mice is followed by accelerated distinct repair mechanisms in Staphylococcus aureus-induced murine brain abscess.

    PubMed

    Held, Josephin; Preuße, Corinna; Döser, Alexandra; Richter, Lydia; Heppner, Frank L; Stenzel, Werner

    2013-09-01

    Murine Staphylococcus aureus-mediated brain abscess comprises 2 major phases, an initial phase of cerebritis, followed by a healing phase characterized by capsule formation. C57BL/6 wild-type (WT) and IL-12p35(-/-) mice were intracerebrally infected with S. aureus to induce brain abscesses. Clinical disease activity and bacterial load were monitored. The cell populations that were involved, as well as their specific mediators, were analyzed by immunohistochemistry, quantitative real-time polymerase chain reaction, and flow cytometry. In the acute phase, IL-12p35(-/-) mice were protected from disease. This was associated with enhanced recruitment of granulocytes, accompanied by upregulated expression of Il17a, Csf2 (which encodes granulocyte-macrophage colony-stimulating factor), Cxcl1, and Cxcl5, as well as increased expression of proinflammatory mediators, including Nos2 (which encodes inducible nitric oxide synthase), Ptgs2 (which encodes cyclooxygenase 2), and Tnf, that were primarily produced by granulocytes and activated microglia/macrophages. Furthermore, mechanisms associated with beneficial wound healing, including an accelerated formation of a fibrous capsule, were demonstrated by prominent VEGF-A production and collagen deposition driven by an earlier onset of T-helper 2 immunity in the absence of interleukin 12 (IL-12). Brain abscess development is orchestrated by IL-12 at different stages of disease. Our data indicate that IL-12 has a nonprotective role in the acute phase and that IL-12 deficiency results in the accelerated formation of a protective capsule during the healing phase, which we consider crucial for early recovery from disease.

  2. Macrophages and Myeloid Dendritic Cells Lose T Cell-Stimulating Function in Simian Immunodeficiency Virus Infection Associated with Diminished IL-12 and IFN-α Production.

    PubMed

    Wonderlich, Elizabeth R; Wu, Wen-Chi; Normolle, Daniel P; Barratt-Boyes, Simon M

    2015-10-01

    Impaired T cell responses are a defining characteristic of HIV infection, but the extent to which altered mononuclear phagocyte function contributes to this defect is unclear. We show that mononuclear phagocytes enriched from rhesus macaque lymph nodes have suppressed ability to stimulate CD4 T cell proliferation and IFN-γ release after acute SIV infection. When individual populations were isolated, myeloid dendritic cells (mDC) and macrophages but not plasmacytoid DC (pDC) had suppressed capacity to stimulate CD4 T cell proliferation, with macrophage function declining as infection progressed. Macrophages, but not pDC or mDC, had suppressed capacity to induce IFN-γ release from CD4 T cells in acute infection, even after stimulation with virus-encoded TLR7/8 ligand. Changes in expression of costimulatory molecules did not explain loss of function postinfection. Conversely, pDC and mDC had marked loss of IFN-α and IL-12 production, respectively, and macrophages lost production of both cytokines. In T cell cocultures without TLR7/8 ligand, macrophages were the primary source of IL-12, which was profoundly suppressed postinfection and correlated with loss of IFN-γ release by T cells. TLR7/8-stimulated pDC, mDC and macrophages all produced IL-12 in T cell cocultures, which was suppressed in chronic infection. Supplementing IL-12 enhanced mDC-driven IFN-γ release from T cells, and IL-12 and IFN-α together restored function in TLR7/8-activated macrophages. These findings reveal loss of macrophage and mDC T cell-stimulating function in lymph nodes of SIV-infected rhesus macaques associated with diminished IL-12 and IFN-α production that may be a factor in AIDS immunopathogenesis.

  3. Modulation of anticancer cytokines IL-2 and IL-12 by melatonin and the other pineal indoles 5-methoxytryptamine and 5-methoxytryptophol in the treatment of human neoplasms.

    PubMed

    Lissoni, P

    2000-01-01

    Lymphocyte number still remains one of the most important immune parameters predicting the prognosis of advanced cancer patients. IL-2 and IL-12 are the main antitumor cytokines in humans, and their effect is modulated by the neuroendocrine system, mainly by the pineal gland through the circadian release of melatonin (MLT) and perhaps that of other indole hormones, such as 5-methoxytryptamine (5-MTT), and 5-methoxytryptophol (5-MTP). MLT has been proven to exert important antitumor immunomodulating effects, whereas the possible immunomodulatory properties of the other pineal indoles are still controversial. In an attempt to better define the pineal neuroendocrine regulation of the anticancer cytokine network, we have evaluated in metastatic solid-tumor patients the effects on lymphocyte number induced by different neuroimmune regimens, consisting of MLT alone (20 mg/day orally in the evening), subcutaneous (s.c.) low-dose IL-2 alone (3 MIU/day in the evening for 6 days/week), s.c. low-dose IL-12 alone (0.5 mcg/kg once/week in the morning), IL-12 plus MLT, IL-2 plus MLT, and IL-2 plus MLT plus 5-MTT (10 mg/day orally in the afternoon) plus 5-MTP (5 mg/day orally at noon). The results showed the following evidence: (1) MLT alone is unable to induce lymphocytosis; (2) MLT significantly enhances IL-2-induced lymphocytosis; (3) IL-12 alone determines lymphocytopenia, which can be reversed by MLT; (4) IL-2 plus IL-12 induces a very pronounced lymphocytosis, which can be further amplified by MLT; (5) a total pineal endocrine replacement therapy with MLT, 5-MTT, and 5-MTP further increases IL-2-induced lymphocytosis with respect to MLT plus IL-2 alone. Therefore, this study confirms that IL-2- and IL-12-dependent anticancer immunity is under a pineal modulation.

  4. [Amino acids 395-416 in DNA binding domain of STAT4 is involved in IL-12-induced nuclear import of STAT4].

    PubMed

    Huang, Yu-Mei; Wen, Ya-Ping; Li, Xuan-An; Yuan, Yuan; Luo, Qi-Zhi; Li, Ming

    2012-08-25

    The purpose of the present study is to explore the mechanism of IL-12-induced nuclear import of signal transducer and activator of transcription 4 (STAT4). Assayed by analyses of homology alignment of STATs, amino acids 395-416 in DNA binding domain was found to be a potential dimer-specific nuclear localization signal (dsNLS) of STAT4. Therefore, several plasmids were constructed. Wild-type STAT4 was inserted into the SalI and BamHI sites of pEGFP-C1 for the construction of plasmid pEGFP-STAT4. The DNA fragment of STAT4 with the deletion of amino acids 395-416 was amplified by RCR and introduced into the SalI and BamHI sites of pEGFP-C1 which was named pEGFP-STAT4-Del. Classic NLS DNA sequence of SV40 T antigen was inserted into the XhoI and HindIII sites of pEGFP-C1. This plasmid was named as pEGFP-NLS and used as a positive control. Plasmid pEGFP-NLS-STAT4-Del was constructed by inserting STAT4-Del into SalI and BamHI sites of pEGFP-NLS. These plasmids were transiently transfected into Caski cells, respectively. The results showed that, after these transfected cells were stimulated by IL-12, wild type STAT4 existed in the cytoplasm at 0 min, and was predominantly localized to the nucleus at 45 min, and distributed in both cytoplasm and nucleus at 60 min, suggesting that STAT4 translocates from cytoplasm into nucleus and finally re-entries into the cytoplasm during the stimulation of IL-12. However, deletion mutant of STAT4 was arrested in cytoplasm during the IL-12 stimulation. Leptomycin B, which specifically blocks protein export from nucleus into cytoplasm, was used to further demonstrate whether STAT4-Del is transferred into nucleus even with stimulation of IL-12. After the transfected cells were pre-treated by leptomycin B, the wild type STAT4 was mainly localized in nucleus after the IL-12 stimulation, suggesting that STAT4 was translocated from cytoplasm into nucleus by the stimulation of IL-12. On the other hand, the deletion mutant of STAT4 distributed

  5. Polymorphisms in genes of interleukin 12 and its receptors and their association with protection against severe malarial anaemia in children in western Kenya.

    PubMed

    Zhang, Lyna; Prather, Donald; Vanden Eng, Jodi; Crawford, Sara; Kariuki, Simon; ter Kuile, Feiko; Terlouw, Dianne; Nahlen, Bernard; Lal, Altaf A; Slutsker, Laurence; Udhayakumar, Venkatachalam; Shi, Ya Ping

    2010-03-29

    Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined. Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya. An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21). This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

  6. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism.

    PubMed

    Kullberg, M C; Ward, J M; Gorelick, P L; Caspar, P; Hieny, S; Cheever, A; Jankovic, D; Sher, A

    1998-11-01

    Mice rendered deficient in interleukin-10 (IL-10) by gene targeting (IL-10(-/-) mice) develop chronic enterocolitis resembling human inflammatory bowel disease (IBD) when maintained in conventional animal facilities. However, they display a minimal and delayed intestinal inflammatory response when reared under specific-pathogen-free (SPF) conditions, suggesting the involvement of a microbial component in pathogenesis. We show here that experimental infection with a single bacterial agent, Helicobacter hepaticus, induces chronic colitis in SPF-reared IL-10(-/-) mice and that the disease is accompanied by a type 1 cytokine response (gamma interferon [IFN-gamma], tumor necrosis factor alpha, and nitric oxide) detected by restimulation of spleen and mesenteric lymph node cells with a soluble H. hepaticus antigen (Ag) preparation. In contrast, wild-type (WT) animals infected with the same bacteria did not develop disease and produced IL-10 as the dominant cytokine in response to Helicobacter Ag. Strong H. hepaticus-reactive antibody responses as measured by Ag-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, IgG3, and IgA were observed in both WT and IL-10(-/-) mice. In vivo neutralization of IFN-gamma or IL-12 resulted in a significant reduction of intestinal inflammation in H. hepaticus-infected IL-10(-/-) mice, suggesting an important role for these cytokines in the development of colitis in the model. Taken together, these microbial reconstitution experiments formally establish that a defined bacterial agent can serve as the immunological target in the development of large bowel inflammation in IL-10(-/-) mice and argue that in nonimmunocompromised hosts IL-10 stimulated in response to intestinal flora is important in preventing IBD.

  7. Broadly impaired NK cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro.

    PubMed

    Johansson, Sofia E; Hall, Håkan; Björklund, Jens; Höglund, Petter

    2004-01-01

    NK cells represent a link between innate and adaptive immunity, and may play a role in regulating autoimmune disorders. We have characterized the NK cell population in non-obese diabetic (NOD) mice. The percentage and absolute numbers of NK cells were similar in NOD and control MHC-matched B6.g7 mice. However, the capacity of NOD NK cells to mediate natural cytotoxicity as well as FcR- and Ly49D-mediated killing was compromised in vitro, suggesting a defect affecting multiple activation pathways. The defect was neither linked to the NK gene complex nor to the MHC, as determined by comparison with mice congenic for these regions. Introducing the beta(2)-microglobulin mutation on the NOD background further impaired NK cell function, showing that the compromised cytotoxic capacity in these two strains arises from two independent mechanisms. In vivo rejection responses against tumor cells and against MHC class I-deficient spleen cells were decreased in naive NOD recipients, but restored in mice pre-activated with tilorone, a potent activator of NK cells. In addition, killing of some tumor targets was restored in vitro after activation of NK cells with IL-12 plus IL-18 or with IFN-alpha/beta, but not with IL-2. Interestingly, natural killing of RMA-S targets by NOD NK cells could not be restored in vitro, indicating that restoration of killing capacity was only partial. Our data suggest a severe, but partially restorable, killing defect in NOD NK cells, affecting activation through several pathways.

  8. IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4(+) T cells.

    PubMed

    Ohno, Yosuke; Kitamura, Hidemitsu; Takahashi, Norihiko; Ohtake, Junya; Kaneumi, Shun; Sumida, Kentaro; Homma, Shigenori; Kawamura, Hideki; Minagawa, Nozomi; Shibasaki, Susumu; Taketomi, Akinobu

    2016-02-01

    Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4(+) T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b(+)CD11c(+) cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b(+)CD11c(+) cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b(+)CD11c(+) cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4(+) T and CD8(+) T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy.

  9. Reciprocal induction of IL-10 and IL-12 from macrophages by low-density lipoprotein and its oxidized forms.

    PubMed

    Varadhachary, A S; Monestier, M; Salgame, P

    2001-10-10

    Atherosclerosis is a chronic inflammatory disease. Several lines of evidence indicate that altered or modified lipoproteins contribute to plaque formation and lesion progression in atherogenesis. In this study we examined if lipoproteins and their oxidized forms can exert an immunomodulatory effect, thereby potentially influencing atherogenesis. We demonstrate that LDL, upon binding to its receptor, induces interleukin (IL)-10 production from macrophages and biases naive T cells to become Th2-like. In contrast, oxLDL induces IL-12 from macrophages and accordingly favors differentiation of naive T cells along a Th1 pathway. IL-10 is a potent anti-inflammatory cytokine with a number of potential effects that could dampen inflammation at sites of vascular wall damage, including downregulation of MHC and adhesion molecules and biasing of adaptive immune responses toward the anti-inflammatory, humoral immune-promoting Th2 T cell subset. These studies assign a new immunomodulatory role to LDLs and offer a potential means to upregulate IL-10 production and prevent arterial inflammation.

  10. Epstein-Barr viral miRNAs inhibit antiviral CD4+ T cell responses targeting IL-12 and peptide processing

    PubMed Central

    Moosmann, Andreas; Mautner, Josef; Zielinski, Christina

    2016-01-01

    Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection. PMID:27621419

  11. Migratory CD103+ dendritic cells suppress helminth-driven type 2 immunity through constitutive expression of IL-12

    PubMed Central

    Tussiwand, Roxane; Dreesen, Leentje; Fairfax, Keke C.; Huang, Stanley Ching-Cheng; Smith, Amber M.; O’Neill, Christina M.; Lam, Wing Y.; Edelson, Brian T.; Urban, Joseph F.; Murphy, Kenneth M.

    2016-01-01

    CD8α+ and CD103+ dendritic cells (DCs) play a central role in the development of type 1 immune responses. However, their role in type 2 immunity remains unclear. We examined this issue using Batf3−/− mice, in which both of these DC subsets are missing. We found that Th2 cell responses, and related events such as eosinophilia, alternative macrophage activation, and immunoglobulin class switching to IgG1, were enhanced in Batf3−/− mice responding to helminth parasites. This had beneficial or detrimental consequences depending on the context. For example, Batf3 deficiency converted a normally chronic intestinal infection with Heligmosomoides polygyrus into an infection that was rapidly controlled. However, liver fibrosis, an IL-13–mediated pathological consequence of wound healing in chronic schistosomiasis, was exacerbated in Batf3−/− mice infected with Schistosoma mansoni. Mechanistically, steady-state production of IL-12 by migratory CD103+ DCs, independent of signals from commensals or TLR-initiated events, was necessary and sufficient to exert the suppressive effects on Th2 response development. These findings identify a previously unrecognized role for migratory CD103+ DCs in antagonizing type 2 immune responses. PMID:26712805

  12. A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo.

    PubMed

    Soares, Helena; Waechter, HaeNa; Glaichenhaus, Nicholas; Mougneau, Evelyne; Yagita, Hideo; Mizenina, Olga; Dudziak, Diana; Nussenzweig, Michel C; Steinman, Ralph M

    2007-05-14

    Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.

  13. Paracoccidioides brasiliensis disseminated disease in a patient with inherited deficiency in the beta1 subunit of the interleukin (IL)-12/IL-23 receptor.

    PubMed

    Moraes-Vasconcelos, Dewton de; Grumach, Anete S; Yamaguti, Augusto; Andrade, Maria Elisa B; Fieschi, Claire; de Beaucoudrey, Ludovic; Casanova, Jean-Laurent; Duarte, Alberto J S

    2005-08-15

    Paracoccidioides brasiliensis is a facultative intracellular dimorphic fungus that causes paracoccidioidomycosis (PCM), the most important deep mycosis in Latin America. Only a small percentage of individuals infected by P. brasiliensis develop clinical PCM, possibly in part because of genetically determined interindividual variability of host immunity. However, no primary immunodeficiency has ever been associated with PCM. We describe the first patient, to our knowledge, with PCM and a well-defined primary immunodeficiency in the beta 1 subunit of the interleukin (IL)-12/IL-23 receptor, a disorder previously shown to be specifically associated with impaired interferon (IFN)-gamma production, mycobacteriosis, and salmonellosis. Our patient had a childhood history of bacille Calmette-Guérin disease and nontyphoid salmonellosis and, at the age of 20 years, presented to our clinic with a disseminated (acute) form of PCM. He responded well to antifungal treatment and is now doing well at 24 years of age. This unique observation supports previous studies of PCM suggesting that IL-12, IL-23, and IFN-gamma play an important role in protective immunity to P. brasiliensis. Tuberculosis and PCM are thus not only related clinically and pathologically, but also by their immunological pathogenesis. Our study further expands the spectrum of clinical manifestations of inherited defects of the IL-12/IL-23-IFN-gamma axis. Patients with unexplained deep fungal infections, such as PCM, should be tested for defects in the IL-12/IL-23-IFN- gamma axis.

  14. Adjuvant Cationic Liposomes Presenting MPL and IL-12 Induce Cell Death, Suppress Tumor Growth, and Alter the Cellular Phenotype of Tumors in a Murine Model of Breast Cancer

    PubMed Central

    2015-01-01

    Dendritic cells (DC) process and present antigens to T lymphocytes, inducing potent immune responses when encountered in association with activating signals, such as pathogen-associated molecular patterns. Using the 4T1 murine model of breast cancer, cationic liposomes containing monophosphoryl lipid A (MPL) and interleukin (IL)-12 were administered by intratumoral injection. Combination multivalent presentation of the Toll-like receptor-4 ligand MPL and cytotoxic 1,2-dioleoyl-3-trmethylammonium-propane lipids induced cell death, decreased cellular proliferation, and increased serum levels of IL-1β and tumor necrosis factor (TNF)-α. The addition of recombinant IL-12 further suppressed tumor growth and increased expression of IL-1β, TNF-α, and interferon-γ. IL-12 also increased the percentage of cytolytic T cells, DC, and F4/80+ macrophages in the tumor. While single agent therapy elevated levels of nitric oxide synthase 3-fold above basal levels in the tumor, combination therapy with MPL cationic liposomes and IL-12 stimulated a 7-fold increase, supporting the observed cell cycle arrest (loss of Ki-67 expression) and apoptosis (TUNEL positive). In mice bearing dual tumors, the growth of distal, untreated tumors mirrored that of liposome-treated tumors, supporting the presence of a systemic immune response. PMID:25179345

  15. IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia.

    PubMed

    Pegram, H J; Purdon, T J; van Leeuwen, D G; Curran, K J; Giralt, S A; Barker, J N; Brentjens, R J

    2015-02-01

    Disease relapse or progression is a major cause of death following umbilical cord blood (UCB) transplantation (UCBT) in patients with high-risk, relapsed or refractory acute lymphoblastic leukemia (ALL). Adoptive transfer of donor-derived T cells modified to express a tumor-targeted chimeric antigen receptor (CAR) may eradicate persistent disease after transplantation. Such therapy has not been available to UCBT recipients, however, due to the low numbers of available UCB T cells and the limited capacity for ex vivo expansion of cytolytic cells. We have developed a novel strategy to expand UCB T cells to clinically relevant numbers in the context of exogenous cytokines. UCB-derived T cells cultured with interleukin (IL)-12 and IL-15 generated >150-fold expansion with a unique central memory/effector phenotype. Moreover, UCB T cells were modified to both express the CD19-specific CAR, 1928z, and secrete IL-12. 1928z/IL-12 UCB T cells retained a central memory-effector phenotype and had increased antitumor efficacy in vitro. Furthermore, adoptive transfer of 1928z/IL-12 UCB T cells resulted in significantly enhanced survival of CD19(+) tumor-bearing SCID-Beige mice. Clinical translation of CAR-modified UCB T cells could augment the graft-versus-leukemia effect after UCBT and thus further improve disease-free survival of transplant patients with B-cell ALL.

  16. TLR4-mediated IL-12 production enhances IFN-γ and IL-1β production, which inhibits TGF-β production and promotes antibody-induced joint inflammation

    PubMed Central

    2012-01-01

    Introduction Toll-like receptor (TLR)4 promotes joint inflammation in mice. Despite that several studies report a functional link between TLR4 and interleukin-(IL-)1β in arthritis, TLR4-mediated regulation of the complicated cytokine network in arthritis is poorly understood. To address this, we investigated the mechanisms by which TLR4 regulates the cytokine network in antibody-induced arthritis. Methods To induce arthritis, we injected mice with K/BxN serum. TLR4-mediated pathogenesis in antibody-induced arthritis was explored by measuring joint inflammation, cytokine levels and histological alteration. Results Compared to wild type (WT) mice, TLR4-/- mice showed attenuated arthritis and low interferon (IFN)-γ, IL-12p35 and IL-1β transcript levels in the joints, but high transforming growth factor (TGF)-β expression. Injection of lipopolysaccharide (LPS) enhanced arthritis and exaggerated joint cytokine alterations in WT, but not TLR4-/- or IL-12p35-/- mice. Moreover, STAT4 phosphorylation in joint cells and intracellular IL-12p35 expression in macrophages, mast cells and Gr-1+ cells were detected in WT mice with arthritis and enhanced by LPS injection. Therefore, IL-12p35 appears to act downstream of TLR4 in antibody-induced arthritis. TLR4-mediated IL-12 production enhanced IFN-γ and IL-1β production via T-bet and pro-IL-1β production. Recombinant IL-12, IFN-γ and IL-1β administration restored arthritis, but reduced joint TGF-β levels in TLR4-/- mice. Moreover, a TGF-β blockade restored arthritis in TLR4-/- mice. Adoptive transfer of TLR4-deficient macrophages and mast cells minimally altered joint inflammation and cytokine levels in macrophage- and mast cell-depleted WT mice, respectively, whereas transfer of WT macrophages or mast cells restored joint inflammation and cytokine expression. Gr-1+ cell-depleted splenocytes partially restored arthritis in TLR4-/- mice. Conclusion TLR4-mediated IL-12 production by joint macrophages, mast cells and Gr-1

  17. Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection

    PubMed Central

    1994-01-01

    Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN- gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in

  18. Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice.

    PubMed

    Gabaglia, Claudia Raja; DeLaney, Alexandra; Gee, Jennifer; Halder, Ramesh; Graham, Frank L; Gauldie, Jack; Sercarz, Eli E; Braciak, Todd A

    2010-10-14

    Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.

  19. [Influence of steroid hormones on the production of two inflammatory markers, IL-12 and nitric oxide, in Behçet's disease].

    PubMed

    Ahmedi, M L; Belguendouz, H; Messaoudene, D; Mesbah-Amroun, H; Terahi, M; Lahlou-Boukoffa, O S; Touil-Boukoffa, C

    2016-04-01

    Uveitis is one of the major diagnostic criteria of Behçet's disease (BD), a chronic systemic inflammatory pathology with an uncertain etiology. Since uveitis is more frequent in male patients, we assessed the level and the effect of sex hormones on inflammatory responses during BD. Peripheral blood was taken from 19 patients with BD and 20 healthy subjects. Estradiol, testosterone and cortisol were measured in plasma by ELISA. Circulating mononuclear cells (PBMC) were obtained on gradient density and cultured with or without the three hormones for 24h at 37 ̊C. IL-12 and nitric oxide (NO) were measured in vivo and ex vivo by ELISA and a modified Griess method, respectively. We confirmed the significantly higher in vivo and in vitro levels of NO and IL-12 in BD in comparison to controls (P<0.05). We also found that circulating cortisol was lower in BD while sex hormones did not show any significant difference between the groups (P>0.05). In vitro, NO was reduced by estradiol and cortisol and increased by testosterone in both sexes. In contrast, while IL-12 production showed the same production profile as NO in women, estradiol and cortisol failed to reduce IL-12 levels in men. Our results may explain in part the differences observed between men and women in disease clinical expression. In fact, male patients seem to have defective IL-12 down-regulation by estradiol and cortisol that increases Th1 immune responses. This may be implicated in the severe expression of BD in men.

  20. Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice

    PubMed Central

    2010-01-01

    Background Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. Methods The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. Results Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. Conclusion Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment. PMID:20946663

  1. Peroxiredoxin 3 promotes IL-12 production from macrophages and partially protects mice against infection with Toxoplasma gondii.

    PubMed

    Fereig, Ragab M; Nishikawa, Yoshifumi

    2016-12-01

    Toxoplasmosis remains a life-threatening infection of humans and various domestic and wild animals worldwide. It is caused by the obligatory intracellular protozoan parasite Toxoplasma gondii. Peroxiredoxins (Prxs) are a family of antioxidant enzymes that protect cells from oxidative stress from hydroperoxides. In the recent years, several studies have reported the potential use of T. gondii-derived enzymes in triggering protective immunity against T. gondii infection. Therefore, this study was conducted to investigate the immunogenicity and protective efficacy of TgPrx3. In vitro stimulation of peritoneal macrophages with recombinant TgPrx3 protein fused to glutathione-S transferase (TgPrx3-GST) enhanced IL-12p40 production, indicating the immune-stimulating potentials of TgPrx3. Next, protective efficacy was investigated by subcutaneous inoculation of mice with TgPrx3-GST (25pmol), and recombinant GST or PBS were used as the controls. Mice immunized with TgPrx3-GST exhibited a significant elevation of specific antibodies in terms of IgG1 and IgG2c isotypes. Moreover, interferon-gamma production and spleen cell proliferation dramatically increased in the TgPrx3-GST-sensitized cells from mice immunized with the same antigen. The severity of the T. gondii infections tended to be attenuated in the TgPrx3-GST-immunized mice, as evidenced by their higher survival rates and lower parasite burdens in the brain. Altogether, TgPrx3 immunization induced specific humoral and cellular immune responses and partially protected the mice against lethal toxoplasmosis. Our results suggest the possible use of TgPrx3 as a vaccine candidate against T. gondii infections. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKKβ-AMPK-SIRT1 Signaling Pathway

    PubMed Central

    Zhu, Yuanfeng; Yang, Yongjun; Chen, Xiaoli; Fan, Shijun; Chen, Qian; Zheng, Jiang

    2016-01-01

    Activated macrophages are the primary sources of IL-12, a key cytokine bridging innate and adaptive immunity. However, macrophages produce low amounts of IL-12 upon stimulation and the underlying regulatory mechanism remains unclear. In this study, we found a new calcium-dependent mechanism that controlled IL-12 production in LPS-treated murine macrophages. First, LPS was demonstrated to induce extracellular calcium entry in murine peritoneal macrophages and inhibition of calcium influx resulted in marked enhancement in IL-12 production. Then, withdrawal of extracellular calcium was found to suppress CaMKKβ and AMPK activation triggered by LPS while chemical inhibition or genetic knockdown of these two kinases augmented LPS induced IL-12 production. AMPK activation increased the NAD+/NADH ratio and activated Sirtuin 1 (SIRT1), a NAD+-dependent deacetylating enzyme and negative regulator of inflammation. Chemical inhibitor or siRNA of SIRT1 enhanced IL-12 release while its agonist suppressed IL-12 production. Finally, it was found that SIRT1 selectively affected the transcriptional activity of NF-κB which thereby inhibited IL-12 production. Overall, our study demonstrates a new role of transmembrane calcium mobilization in immunity modulation such that inhibition of calcium influx leads to impaired activation of CaMKKβ-AMPK-SIRT1 signaling pathway which lifts restriction on NF-κB activation and results in enhanced IL-12 production. PMID:27313401

  3. An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells

    PubMed Central

    Naegelen, Isabelle; Plançon, Sébastien; Nicot, Nathalie; Kaoma, Tony; Muller, Arnaud; Vallar, Laurent; Tschirhart, Eric J.; Bréchard, Sabrina

    2015-01-01

    Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. PMID:25548252

  4. Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma.

    PubMed

    Sedlar, Ales; Kranjc, Simona; Dolinsek, Tanja; Cemazar, Maja; Coer, Andrej; Sersa, Gregor

    2013-01-29

    Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD(50) assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.

  5. Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

    PubMed Central

    2013-01-01

    Background Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. Methods Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. Results Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. Conclusions Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation. PMID:23360213

  6. Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells

    PubMed Central

    Ruffell, Brian; Chang-Strachan, Debbie; Chan, Vivien; Rosenbusch, Alexander; Ho, Christine M.T.; Pryer, Nancy; Daniel, Dylan; Hwang, E. Shelley; Rugo, Hope S.; Coussens, Lisa M.

    2014-01-01

    Summary Blockade of colony-stimulating factor-1 (CSF-1) limits macrophage infiltration and improves response of mammary carcinomas to chemotherapy. Herein we identify interleukin (IL)-10 expression by macrophages as the critical mediator of this phenotype. Infiltrating macrophages were the primary source of IL-10 within tumors, and therapeutic blockade of IL-10 receptor (IL-10R) was equivalent to CSF-1 neutralization in enhancing primary tumor response to paclitaxel and carboplatin. Improved response to chemotherapy was CD8+ T cell-dependent, however IL-10 did not directly suppress CD8+ T cells or alter macrophage polarization. Instead, IL-10R blockade increased intratumoral dendritic cell expression of IL-12, which was necessary for improved outcomes. In human breast cancer, expression of IL12A and cytotoxic effector molecules were predictive of pathological complete response rates to paclitaxel. PMID:25446896

  7. Mycobacterium indicus pranii (Mw) Re-Establishes Host Protective Immune Response in Leishmania donovani Infected Macrophages: Critical Role of IL-12

    PubMed Central

    Adhikari, Anupam; Gupta, Gaurav; Majumder, Saikat; Banerjee, Sayantan; Bhattacharjee, Surajit; Bhattacharya, Parna; Kumari, Sangeeta; Haldar, Subhadra; Majumdar, Suchandra Bhattacharyya; Saha, Bhaskar; Majumdar, Subrata

    2012-01-01

    Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects. PMID:22792256

  8. Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors

    PubMed Central

    Belderbos, Mirjam E.; Levy, Ofer; Stalpers, Femke; Kimpen, Jan L.; Meyaard, Linde; Bont, Louis

    2012-01-01

    Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection. PMID:22442690

  9. Lymphadenitis caused by infection with an isoniazid- and rifampin-resistant strain of Mycobacterium bovis BCG in an infant with IFN-γ/IL-12 pathway defect*

    PubMed Central

    Diniz, Lilian Martins Oliveira; Guimarães, Tiago; de Oliveira, Maria das Graças Rodrigues; Pinto, Jorge Andrade; de Miranda, Silvana Spindola

    2014-01-01

    We report a rare case in a female infant (age, 3.5 months) with primary immunodeficiency (IFN-γ/IL-12 pathway defect) who presented with suppurative lymphadenitis after Mycobacterium bovis BCG vaccination. The strain of M. bovis BCG identified was found to be resistant to isoniazid and rifampin. The patient was treated with a special pharmacological regimen involving isoniazid (in a limited, strategic manner), ethambutol, streptomycin, and IFN-γ, after which there was complete resolution of the lesions. PMID:24831405

  10. Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation.

    PubMed

    Wang, Sen; Chen, Zhiao; Hu, Conghua; Qian, Fangxing; Cheng, Yuming; Wu, Min; Shi, Bisheng; Chen, Jieliang; Hu, Yunwen; Yuan, Zhenghong

    2013-05-15

    It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK-MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

  11. Differential expression of the inflammation marker IL12p40 in the at-risk mental state for psychosis: a predictor of transition to psychotic disorder?

    PubMed

    Föcking, Melanie; Dicker, Patrick; Lopez, Lorna M; Cannon, Mary; Schäfer, Miriam R; McGorry, Patrick D; Smesny, Stefan; Cotter, David R; Amminger, G Paul

    2016-09-20

    The identification of biomarkers of transition from the at-risk mental state (ARMS) to psychotic disorder is important because early treatment of psychosis is associated with improved outcome. Increasing evidence points to an inflammatory contribution to psychosis. We questioned whether raised levels of plasma inflammatory markers predict transition from ARMS to psychotic disorder and whether any such predictors could be reduced by omega-3 (ω-3) polyunsaturated fatty acids (PUFAs). We measured the levels of 40 neuroinflammation biomarkers using a commercially available immunoassay kit. Firstly, we compared inflammatory markers in subjects in the ARMS who transitioned to psychotic disorder (n = 11) compared to subjects who did not (n = 28). Then we compared inflammatory markers in all subjects before and after ω-3 PUFA treatment (n = 40). Our data provides preliminary evidence that elevations in the baseline plasma levels of the inflammatory marker IL12/IL23p40 are associated with transition from ARMS to psychotic disorder. IL12/IL23p40 levels did not change following 12 weeks administration of ω-3 PUFAs. These findings provide evidence that elevated plasma IL12/IL23p40 is a potential biomarker of increased risk for transition to psychotic disorder. Further studies are required to confirm and extend this finding. Our results do not provide support for the possibility that administration of ω-3 PUFAs act to reduced transition to psychotic disorder by reducing blood levels of IL12/IL23p40. ClinicalTrials.gov, a service of the U.S. National Institutes of Health, Identifier: NCT00396643 , last updated December 20, 2007. Retrospectively registered.

  12. IL-2 Suppression of IL-12p70 by a Recombinant HSV-1 Expressing IL-2 Induces T-Cell Auto-Reactivity and CNS Demyelination

    PubMed Central

    Zandian, Mandana; Mott, Kevin R.; Allen, Sariah J.; Chen, Shuang; Arditi, Moshe; Ghiasi, Homayon

    2011-01-01

    To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2rα) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8+ and CD4+ T cells contribute to HSV-IL-2-induced CNS demyelination with CD8+ T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS. PMID:21364747

  13. Intestinal tuberculosis complicated with perforation during anti-tuberculous treatment in a 13-year-old girl with defective mitogen-induced IL-12 production.

    PubMed

    Law, Siu-Tong; Chiu, Sin-Chuen; Li, Kin Kong

    2014-10-01

    Interleukin-12 (IL-12) is a cytokine which is secreted by activated phagocytes and dendritic cells and promotes cell-mediated immunity to intracellular pathogens, by inducing type 1 helper T cell (TH1) responses and interferon- γ (IFN- γ) production. Defects in the IL-12 may cause selective susceptibility to intracellular pathogens, such as mycobacteria. We herein report on a 13-year-old girl with defective mitogen-induced IL-12 production, who developed intestinal tuberculosis with wide dissemination involving the lung and urinary tract. She improved gradually, but developed terminal ileal perforation approximately 6.1 months following initiation of anti-tuberculous treatment. The paradoxical response phenomenon was suspected. The girl subsequently underwent surgical resection of the affected bowel segment with a temporary double barrel stoma, and ileocolonic anastomosis was performed after the completion of the anti-tuberculous therapy. The patient remained well, with no evidence of recurrent tuberculosis in the past 5 years. This case illustrates the possibility of underlying primary immunodeficiency in a patient with disseminated tuberculosis; delayed tuberculous intestinal perforation can develop during chemotherapy for tuberculosis. Copyright © 2012. Published by Elsevier B.V.

  14. CpG-induced antitumor immunity requires IL-12 in expansion of effector cells and down-regulation of PD-1.

    PubMed

    Yin, Peng; Liu, Xin; Mansfield, Aaron S; Harrington, Susan M; Li, Yinghua; Yan, Yiyi; Dong, Haidong

    2016-10-25

    CpG oligodeoxynucleotides, as a ligand of toll-like receptor (TLR)-9, have demonstrated promising antitumor effects in some clinical trials; however, its toxicity and low efficacy as a systemic therapy has limited its therapeutic applications. In order to improve its therapeutic efficacy, we investigated the mechanisms of CpG-induced antitumor immunity in the context of CD8+ T cell responses. We show that IL-12 is required for the expansion of IFN-γ producing tumor-reactive CD8+ T cells capable of rejecting tumors. In addition, CpGs reduced PD-1 expression by effector CD8+ T cells via the IL-12 pathway. The combination of CpG and PD-1 blockade show a synergistic effect in generation of systemic antitumor immunity. Our studies define a critical role of IL-12 in CpG-induced antitumor immunity and provide a rationale for combined therapy with TLR agonists and immune checkpoint blockade in cancer treatment.

  15. Release of IL-12 by dendritic cells activated by TLR ligation is dependent on MyD88 signaling, whereas TRIF signaling is indispensable for TLR synergy.

    PubMed

    Krummen, Mathias; Balkow, Sandra; Shen, Limei; Heinz, Stefanie; Loquai, Carmen; Probst, Hans-Christian; Grabbe, Stephan

    2010-07-01

    Recently, it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study, we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules, thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines, particularly IL-12, whereas the expression of costimulatory molecules remained unchanged. Consequently, synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore, we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However, noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally, we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment, suggesting an autocrine action of type I IFNs.

  16. IL-18, but not IL-12, induces production of IFN-γ in the immunosuppressive environment of HPV16 E7 transgenic hyperplastic skin.

    PubMed

    Gosmann, Christina; Frazer, Ian H; Mattarollo, Stephen R; Blumenthal, Antje

    2014-10-01

    IFN-γ has a central role in the defense against infections and cancer. More recently, however, IFN-γ has also been reported to have immunosuppressive effects in models of autoimmune disease, melanoma, and premalignant skin disease. Although IL-12 and IL-18 are critical inducers of IFN-γ during infection, the mechanisms that induce IFN-γ in an immunosuppressive context are unknown. Previously, we identified a key role for IFN-γ in mediating the suppression of antigen-specific immune responses in a transgenic mouse model of human papillomavirus (HPV)-associated epidermal hyperplasia, driven by the expression of the HPV16 E7 oncoprotein from a keratin 14 promoter (K14E7). We now demonstrate elevated production of IFN-γ, IL-18, and IL-12 by K14E7 transgenic skin compared with nontransgenic skin. IFN-γ in K14E7 transgenic skin was produced predominantly by CD8(+) and CD4(+) T cells, which were present in greater numbers in K14E7 transgenic skin. Production of IFN-γ in K14E7 skin required IL-18 but not IL-12. Our findings show that IL-18 contributes to inducing IFN-γ in an immunosuppressive cutaneous environment caused by viral oncogene-driven hyperplasia.

  17. Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

    SciTech Connect

    Dahlberg, Angela; Auble, Mark R.; Petro, Thomas M. . E-mail: tpetro@unmc.edu

    2006-09-30

    Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-{beta} than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-{gamma}/LPS, but not IFN-{gamma} alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.

  18. A Novel Mechanism Bridging Innate and Adaptive Immunity: IL-12 Induction of CD25 to Form High Affinity IL-2 Receptors on NK Cells

    PubMed Central

    Lee, Seung-Hwan; Fragoso, Maria F.; Biron, Christine A.

    2012-01-01

    NK cell expression and use of the IL-2Rα chain (CD25), required for the high affinity IL-2R, remain poorly understood. The studies reported here demonstrate that infections with MCMV, but not lymphocytic choriomeningitis virus, induce CD25 on NK cells along with high levels of IL-12 and IL-18. The cytokines act ex vivo to elevate CD25 levels, and IL-12 along with the IL-12R and STAT4, but not the NK activating receptor Ly49H, are required for peak induction in vivo. All examined NK cell populations are driven into proliferation and incorporate BrdU in response to high, but only those from MCMV infection respond to low ex vivo concentrations of IL-2. Numbers of NK cells are reduced during MCMV infection by IL-2 neutralization. Thus, a link between innate and adaptive immunity is established by which composition of innate cytokine responses sets up to promote NK cell use of a factor supporting adaptive responses. PMID:22888135

  19. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-04-30

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  20. An Excess of the Pro-inflammatory cytokines IFNγ and IL12 Impairs the Development of the Memory CD8+ T cell Response to Chlamydia trachomatis

    PubMed Central

    Zhang, Xuqing; Starnbach, Michael N.

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Transfer of cultured Chlamydia-specific CD8+ T cells or vaccination with recombinant virus expressing a MHCI-restricted Chlamydia antigen confers protection, yet surprisingly a protective CD8+ T cell response is not stimulated following natural infection. In this study we demonstrate that the presence of excess IL12 and IFNγ contributes to poor memory CD8+ T cell development during C. trachomatis infection of mice. IL12 is required for CD8+ T cell expansion but drives effector CD8+ T cells into a short-lived fate whereas IFNγ signaling impairs the development of effector memory cells. We show that transient blockade of IL12 and IFNγ during priming promotes the development of memory precursor effector CD8+ T cells and increases the number of memory T cells that participate in the recall protection against subsequent infection. Overall, this study identifies key factors shaping memory development of Chlamydia-specific CD8+ T cells that will inform future vaccine development against this and other pathogens. PMID:26179901

  1. Blood dendritic cell levels associated with impaired IL-12 production and T-cell deficiency in patients with kidney disease: implications for post-transplant viral infections.

    PubMed

    Chen, Ping; Sun, Qianmei; Huang, Yanfei; Atta, Mohamed G; Turban, Sharon; Segev, Dorry L; Marr, Kieren A; Naqvi, Fizza F; Alachkar, Nada; Kraus, Edward S; Womer, Karl L

    2014-10-01

    Reduced pretransplant blood myeloid dendritic cell (mDC) levels are associated with post-transplant BK viremia and cytomegalovirus (CMV) disease after kidney transplantation. To elucidate potential mechanisms by which mDC levels might influence these outcomes, we studied the association of mDC levels with mDC IL-12 production and T-cell level/function. Peripheral blood (PB) was studied in three groups: (i) end stage renal disease patients on hemodialysis (HD; n = 81); (ii) chronic kidney disease stage IV-V patients presenting for kidney transplant evaluation or the day of transplantation (Eval/Tx; n = 323); and (iii) healthy controls (HC; n = 22). Along with a statistically significant reduction in mDC levels, reduced CD8(+) T-cell levels were also demonstrated in the kidney disease groups compared with HC. Reduced PB mDC and monocyte-derived DC (MoDC) IL-12 production was observed after in vitro LPS stimulation in the HD versus HC groups. Finally, ELISpot assays demonstrated less robust CD3(+) INF-γ responses by MoDCs pulsed with CMV pp65 peptide from HD patients compared with HC. PB mDC level deficiency in patients with kidney disease is associated with deficient IL-12 production and T-cell level/function, which may explain the known correlation of CD8(+) T-cell lymphopenia with deficient post-transplant antiviral responses. © 2014 Steunstichting ESOT.

  2. Increased Production of IL-4 and IL-12p40 from Bronchoalveolar Lavage Cells Are Biomarkers of Mycobacterium tuberculosis in the Sputum

    PubMed Central

    Nolan, Anna; Fajardo, Elaine; Huie, Maryann L.; Condos, Rany; Pooran, Anil; Dawson, Rodney; Dheda, Keertan; Bateman, Eric; Rom, William N.; Weiden, Michael D.

    2013-01-01

    Background Tuberculosis (TB) causes 1.45 million deaths annually world wide, the majority of which occur in the developing world. Active TB disease represents immune failure to control latent infection from airborne spread. Acid-fast bacillus (AFB) seen on sputum smear is a biomarker for contagiousness. Methods We enrolled 73 tuberculosis patients with extensive infiltrates into a research study using bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production. All patients had sputum culture demonstrating Mycobacterium tuberculosis and 59/73 (81%) had AFB identified by microscopy of the sputum. Compared with smear negative patients, smear positive patients at presentation had a higher proportion with smoking history, a higher proportion with temperature >38.50 C, higher BAL cells/ml, lower percent lymphocytes in BAL, higher IL-4 and IL-12p40 in BAL cell supernatants. There was no correlation between AFB smear and other BAL or serum cytokines. Increasing IL-4 was associated with BAL PMN and negatively associated with BAL lymphocytes. Each 10-fold increase in BAL IL-4 and IL-12p40 increased the odds of AFB smear positivity by 7.4 and 2.2-fold, respectively, in a multi-variable logistic model. Conclusion Increasing IL-4 and IL-12p40 production by BAL cells are biomarkers for AFB in sputum of patients who present with radiographically advanced TB. They likely reflect less effective immune control of pathways for controlling TB, leading to patients with increased infectiousness. PMID:23527200

  3. Combined IL-15 and IL-12 drives the generation of CD34(+)-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer.

    PubMed

    Cany, Jeannette; van der Waart, Anniek B; Spanholtz, Jan; Tordoir, Marleen; Jansen, Joop H; van der Voort, Robbert; Schaap, Nicolaas M; Dolstra, Harry

    2015-07-01

    Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34(+) hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

  4. Antitumor effect of interleukin (IL)-12 in the absence of endogenous IFN-gamma: a role for intrinsic tumor immunogenicity and IL-15.

    PubMed

    Gri, Giorgia; Chiodoni, Claudia; Gallo, Elena; Stoppacciaro, Antonella; Liew, Foo Y; Colombo, Mario P

    2002-08-01

    IFN-gamma knockout mice (GKO) rejected C26 colon carcinoma cells transduced to secrete interleukin(IL)-12 but do not reject similarly transduced TSA mammary adenocarcinoma (C26/12 and TSA/12 cells, respectively). To determine whether such difference could be because of a different tumor response to IFN-gamma, we injected BALB/c mice with TSA, C26, and their IL-12-transduced counterparts rendered unresponsive to IFN-gamma by stable transduction of a dominant negative (DN), truncated IFN-gamma receptor alpha chain. TSA/DN and C26/DN showed the same in vivo growth kinetics as parental cells, whereas coexpression of IL-12 induced rejection independent of tumor-cell responsiveness to IFN-gamma. This suggests that the role of IFN-gamma is primarily in activating the host immune response, which appears to depend on the intrinsic immunogenicity of the target tumor. C26 and TSA share a common tumor-associated antigen, yet C26 cells are more immunogenic than TSA. C26/12 expressed 10-fold higher levels of class I MHC molecules and induced higher CTL activity compared with TSA/12 cells in GKO mice. Moreover, whereas in GKO mice the TSA/12 tumor was associated with a greater number of infiltrating T cells, only those infiltrating C26/12 tumor expressed the activation marker OX40. The search for cytokine(s) that might contribute in determining the different T-cell response to these IL-12-transduced tumors in GKO mice revealed a role of IL-15. In situ hybridization showed IL-15 expression in C26/12 but not in TSA/12 tumors. In addition, injection of GKO mice with soluble IL-15 receptor-alpha to block IL-15 expression prevented rejection of C26/12 cells. Together, the results suggest that in the absence of IFN-gamma, IL-12 can exert antitumor activity through alternative mechanisms, depending on the tumor cell type and the availability of cytokines that can replace IFN-gamma in sustaining T-cell functions.

  5. Genetic variants in IL12 influence both hepatitis B virus clearance and HBV-related hepatocellular carcinoma development in a Chinese male population.

    PubMed

    Tan, Aihua; Gao, Yong; Yao, Ziting; Su, Shining; Jiang, Yonghua; Xie, Yuanliang; Xian, Xiaoying; Mo, Zengnan

    2016-05-01

    IL12 plays a major role not only in inducing appropriate immune responses against viral infections (including HBV) but also in the antitumor immune response. This study was conducted to investigate the relationships of genetic variants in IL12 with hepatitis B virus (HBV) clearance and development of HBV-related hepatocellular carcinoma (HCC). We genotyped three single nucleotide polymorphisms (SNPs) of the IL12A (rs568406 and rs2243115) and IL12B (rs3212227) in 395 HBV-positive HCC patients, 293 persistent HBV carriers and 686 subjects with HBV natural clearance from southern China, using the improved multiplex ligase detection reaction (iMLDR) method. Logistic regression analysis adjusted for age, smoking, and alcohol consumption status showed that rs568408 variant genotypes were significantly associated with host HBV-related HCC risk when compared with persistent HBV carriers, and carriers of the GA + AA genotype decreased the HCC risk in comparison with GG carriers (adjusted OR = 0.53, 95 % CI 0.35-0.80, P = 0.002). No relationships between the rs2243115 and rs3212227 SNPs and HCC risk were observed (all P > 0.05). Besides, rs568408 showed an approaching significant effect on susceptibility to HBV persistent infection (adjusted OR = 1.34, 95 % CI 0.99-1.81, P = 0.057 in dominant genetic models). Furthermore, the TG haplotype was observed to be associated with a significantly increased risk of HBV-related HCC (OR = 1.42, 95 % CI 1.10-1.83, P = 0.006), while TA haplotype was associated with a decreased risk of HBV-related HCC (OR = 0.61, 95 % CI 0.45-0.83, P = 0.002). Our results reveal that the IL12A rs568408 variant may be a marker SNP for risk of both HBV clearance and HBV-related HCC development.

  6. Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells

    PubMed Central

    Spelmink, Laura; Sender, Vicky; Hentrich, Karina; Kuri, Thomas; Plant, Laura

    2016-01-01

    ABSTRACT A functional immune response is crucial to prevent and limit infections with Streptococcus pneumoniae. Dendritic cells (DCs) play a central role in orchestrating the adaptive and innate immune responses by communicating with other cell types via antigen presentation and secretion of cytokines. In this study, we set out to understand how pneumococci activate human monocyte-derived DCs to produce interleukin-12 (IL-12) p70, an important cytokine during pneumococcal infections. We show that IL-12p70 production requires uptake of bacteria as well as the presence of the adaptor molecule TRIF, which is known to transfer signals of Toll-like receptor 3 (TLR3) or TLR4 from the endosome into the cell. While TLR4 is redundant for IL-12p70 production in DCs, we found that TLR3 is required to induce full IL-12p70 secretion. Influenza A virus (IAV) infection of DCs did not induce IL-12p70 but markedly upregulated TLR3 expression that during coinfection with S. pneumoniae significantly enhanced IL-12p70 secretion. Finally, we show that pneumococcal RNA can act as a bacterial stimulus for TLR3 and that it is a key signal to induce IL-12p70 production during challenge of DCs with pneumococci. PMID:26956584

  7. A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation

    PubMed Central

    Wang, Xin; Luo, Cheng; Yu, Dongmei; Wang, Yuheng; Chen, Yucong; Lei, Wen; Gao, Xiangdong; Yao, Wenbing

    2015-01-01

    Interleukin (IL)-12 and IL-23 respectively driving polarization of T helper (Th) 1 and Th17 cells has been strongly implicated in the pathogenesis of both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we first constructed, expressed and purified a novel human truncated IL12rβ1-Fc fusion protein (tIL12rβ1/Fc) binding multiple forms of the p40 subunit of human IL-12 and IL-23. tIL12rβ1/Fc was found to effectively ameliorate MOG35–55-induced EAE through reducing the production of Th1- and Th17-polarized pro-inflammatory cytokines and suppressing inflammation and demyelination in the focused parts. Moreover, tIL12rβ1/Fc suppressed Th1 (IFN-γ+ alone) and IFN-γ+ IL-17+ as well as the population of classic Th17 (IL-17+ alone) cells in vivo. Furthermore, tIL12rβ1/Fc ameliorated EAE at the peak of disease via the inhibition of STAT pathway, thereby causing a prominent reduction of RORγt (Th17) and T-bet (Th1) expression. Notably, tIL12rβ1/Fc could increase the relative number of CD4+ Foxp3+ regulatory T cells. These findings indicates that tIL12rβ1/Fc is a novel fusion protein for specific binding multiple forms of p40 subunit to exert potent anti-inflammatory effects and provides a valuable approach for the treatment of MS and other autoimmune diseases. PMID:26384304

  8. Lipoxin A₄ and 15-epi-lipoxin A₄ protect against experimental cerebral malaria by inhibiting IL-12/IFN-γ in the brain.

    PubMed

    Shryock, Nathaniel; McBerry, Cortez; Salazar Gonzalez, Rosa Maria; Janes, Steven; Costa, Fabio T M; Aliberti, Julio

    2013-01-01

    Cerebral malaria is caused by infection with Plasmodium falciparum and can lead to severe neurological manifestations and predominantly affects sub-Saharan African children. The pathogenesis of this disease involves unbalanced over-production of pro-inflammatory cytokines. It is clear that signaling though IL-12 receptor is a critical step for development of cerebral malaria, IL-12 genetic deficiency failed to show the same effect, suggesting that there is redundancy among the soluble mediators which leads to immunopathology and death. Consequently, counter-regulatory mediators might protect the host during cerebral malaria. We have previously showed that endogenously produced lipoxins, which are anti-inflammatory mediators generated by 5-lipoxygenase (5-LO)-dependent metabolism of arachidonic acid, limit host damage in a model of mouse toxoplasmosis. We postulated here that lipoxins might also play a counter-regulatory role during cerebral malaria. To test this hypothesis, we infected 5-LO-deficient hosts with P. berghei ANKA strain, which induces a mouse model of cerebral malaria (ECM). Our results show accelerated mortality concomitant with exuberant IL-12 and IFN-γ production in the absence of 5-lipoxygenase. Moreover, in vivo administration of lipoxin to 5-LO-deficient hosts prevented early mortality and reduced the accumulation of CD8(+)IFN-γ (+) cells in the brain. Surprisingly, WT animals treated with lipoxin either at the time of infection or 3 days post-inoculum also showed prolonged survival and diminished brain inflammation, indicating that although protective, endogenous lipoxin production is not sufficient to optimally protect the host from brain damage in cerebral malaria. These observations establish 5-LO/LXA4 as a host protective pathway and suggest a new therapeutic approach against human cerebral malaria (HCM). (255 words).

  9. Human epidermal Langerhans cells differ from monocyte-derived Langerhans cells in CD80 expression and in secretion of IL-12 after CD40 cross-linking.

    PubMed

    Peiser, Matthias; Wanner, Reinhard; Kolde, Gerhard

    2004-09-01

    Langerhans cells (LCs) represent an immature population of myeloid dendritic cells (DCs). As a result of their unique Birbeck granules (BGs), langerin expression, and heterogeneous maturation process, they differ from other immature DCs. Monocyte-derived LCs (MoLCs) mimic epidermal LCs. MoLCs with characteristic BGs are generated by culturing blood-derived monocytes with granulocyte macrophage-colony stimulating factor, interleukin (IL)-4, and transforming growth factor-beta1. Here, we compare maturation-induced antigen expression and cytokine release of LCs with MoLCs. To achieve comparable cell populations, LCs and MoLCs were isolated by CD1c cell sorting, resulting in high purity. In unstimulated cells, CD40 was expressed at equal levels. After stimulation with CD40 ligand (CD40L), LCs and MoLCs acquired CD83 and increased CD86. High CD80 expression was exclusively detected in CD1c-sorted MoLCs. Human leukocyte antigen-DR and CD54 expression was found in all cell populations, however, at different intensities. CD40 triggering increased the potency of LCs and MoLCs to stimulate CD4+ T cell proliferation. Activated MoLCs released IL-12p70 and simultaneously, anti-inflammatory IL-10. The application of the Toll-like receptor ligands peptidoglycan, flagellin, and in particular, lipopolysaccharide (LPS) increased the corelease of these cytokines. LCs secreted IL-10 at a comparable level with MoLCs but failed to produce high amounts of IL-12p70 after application of danger signals. These data indicate that MoLCs as well as LCs display no maturation arrest concerning CD83 and CD86 expression. In difference to MoLCs, LCs resisted activation by CD40L and LPS in terms of IL-12 production. This shows that natural and generated LCs share similar features but differ in relevant functions.

  10. Myeloid-Restricted AMPKα1 Promotes Host Immunity and Protects against IL-12/23p40-Dependent Lung Injury during Hookworm Infection.

    PubMed

    Nieves, Wildaliz; Hung, Li-Yin; Oniskey, Taylor K; Boon, Louis; Foretz, Marc; Viollet, Benoit; Herbert, De'Broski R

    2016-06-01

    How the metabolic demand of parasitism affects immune-mediated resistance is poorly understood. Immunity against parasitic helminths requires M2 cells and IL-13, secreted by CD4(+) Th2 and group 2 innate lymphoid cells (ILC2), but whether certain metabolic enzymes control disease outcome has not been addressed. This study demonstrates that AMP-activated protein kinase (AMPK), a key driver of cellular energy, regulates type 2 immunity and restricts lung injury following hookworm infection. Mice with a selective deficiency in the AMPK catalytic α1 subunit in alveolar macrophages and conventional dendritic cells produced less IL-13 and CCL17 and had impaired expansion of ILC2 in damaged lung tissue compared with wild-type controls. Defective type 2 responses were marked by increased intestinal worm burdens, exacerbated lung injury, and increased production of IL-12/23p40, which, when neutralized, restored IL-13 production and improved lung recovery. Taken together, these data indicate that defective AMPK activity in myeloid cells negatively impacts type 2 responses through increased IL-12/23p40 production. These data support an emerging concept that myeloid cells and ILC2 can coordinately regulate tissue damage at mucosal sites through mechanisms dependent on metabolic enzyme function.

  11. Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12

    PubMed Central

    Janelsins, Brian M.; Sumpter, Tina L.; Tkacheva, Olga A.; Rojas-Canales, Darling M.; Erdos, Geza; Mathers, Alicia R.; Shufesky, William J.; Storkus, Walter J.; Falo, Louis D.; Morelli, Adrian E.; Larregina, Adriana T.

    2013-01-01

    Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses. PMID:23365459

  12. IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8+ T cells.

    PubMed

    Stark, Regina; Hartung, Anett; Zehn, Dietmar; Frentsch, Marco; Thiel, Andreas

    2013-06-01

    CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

  13. Tim-3 alters the balance of IL-12/IL-23 and drives TH17 cells: role in hepatitis B vaccine failure during hepatitis C infection

    PubMed Central

    Wang, Jia M.; Ma, Cheng J.; Li, Guang Y.; Wu, Xiao Y.; Thayer, Penny; Greer, Pamela; Smith, Ashley M.; High, Kevin P.; Moorman, Jonathan P; Yao, Zhi Q.

    2013-01-01

    Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production with in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection. PMID:23499521

  14. Protein from intestinal Eimeria protozoan stimulates IL-12 release from dendritic cells, exhibits antitumor properties in vivo and is correlated with low intestinal tumorigenicity.

    PubMed

    Rosenberg, Barnett; Juckett, David A; Aylsworth, Charles F; Dimitrov, Nikolay V; Ho, Siu-Cheong; Judge, John W; Kessel, Sarah; Quensen, Janet; Wong, Kwai-Pheng Ho; Zlatkin, Igor; Zlatkin, Tanya

    2005-05-01

    The small intestine (SI) of vertebrates exhibits low tumorigenesis and rarely supports metastatic growth from distant tumors. Many theories have been proposed to address this phenomenon, but none has been consistently supported. One candidate mechanism is that the vast immunologic compartment of the SI provides a heightened level of tumor immunosurveillance. Consistent with this, we have identified a molecule of low abundance from bovine SI that has the hallmarks of a potent immunostimulant and may be associated with the natural suppression of cancer in the intestinal tract. The protein originates from an endemic gut protozoan, Eimeria spp., and is homologous to the antigen 3-1E previously isolated from the avian apicomplexan E. acervulina. We show here that it is a very potent stimulator of IL-12 release from dendritic cells, upregulates inflammatory modulators in vivo (IL-12, MCP-1, IL-6, TNF-alpha and INF-gamma) and has antitumor properties in mice. In addition, it is synergistic in vitro with anti-CD40 antibody, IFN-gamma, IL-4 and GM-CSF; is active across species barriers in vivo; and has no observable toxicity. Based on these activities, we speculate that it is an inducer of protozoan-targeted innate immunity, which may explain its potential benefit to the intestinal tract and potency as an agent in cancer immunotherapy.

  15. Elevated IL-3 and IL-12p40 levels in the lower airway of infants with RSV-induced bronchiolitis correlate with recurrent wheezing.

    PubMed

    Bertrand, Pablo; Lay, Margarita K; Piedimonte, Giovanni; Brockmann, Pablo E; Palavecino, Christian E; Hernández, Jury; León, Miguel A; Kalergis, Alexis M; Bueno, Susan M

    2015-12-01

    Respiratory Syncytial Virus (RSV) is the first cause of hospitalization due to bronchiolitis in infants. RSV bronchiolitis has been linked to asthma and recurrent wheezing, however the mechanisms behind this association have not been elucidated. Here, we evaluated the cytokine and chemokine profiles in the airways in infants with RSV bronchiolitis. Nasopharyngeal Aspirates (NPA) and Bronchoalveolar Lavage Fluids (BALF) from infants hospitalized due to RSV bronchiolitis and healthy controls were analyzed for cytokine and chemokine production. We observed elevated levels of Th2 cytokines (IL-3, IL-4, IL-10 and IL-13), pro-inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-β, MCP-1/CCL2, MIP-1α/CCL3 and IL-8/CXCL8) in BALF from infants with RSV bronchiolitis, as compared to controls. We found a direct correlation of IL-3 and IL-12p40 levels with the development of recurrent wheezing later in life. These results suggest that IL-3 and IL-12p40 could be considered as molecular predictors for recurrent wheezing due to RSV infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Dual TNF-α/IL-12p40 Interference as a Strategy to Protect Against Colitis Based on miR-16 Precursors With Macrophage Targeting Vectors.

    PubMed

    Huang, Zhen; Ma, Junting; Chen, Mengjie; Jiang, Haoyang; Fu, Yong; Gan, Jingjing; Dong, Lei; Zhang, Junfeng; Chen, Jiangning

    2015-10-01

    Cytokines are central components of the mucosal inflammatory responses that take place during the development of Crohn's disease. Cell-specific combination therapies against cytokines may lead to increased efficacy and even reduced side effects. Therefore, a colonic macrophage-specific therapy using miR-16 precursors that can target both TNF-α and IL-12p40 was tested for its efficacy in experimental colitic mice. Galactosylated low molecular weight chitosan (G-LMWC) associated with miR-16 precursors were intracolonically injected into mice. The cellular localization of miR-16 precursors was determined. The therapeutic effects and possible mechanism were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. The results show that specific upregulation of miR-16 level in colonic macrophages significantly reduces TNF-α and IL-12p40 expression, which could suppress the associated mucosal inflammation and ultimately result in the relief of colitic symptoms. This strategy, based on the dual silencing of colonic macrophage-specific cytokines, represents a potential therapeutic approach that may be valuable for colitis therapy.

  17. Effect of HSV-IL12 Loaded Tumor Cell-Based Vaccination in a Mouse Model of High-Grade Neuroblastoma

    PubMed Central

    Pereboeva, Larisa; Gillespie, G. Yancey; Cloud, Gretchen A.; Langford, Catherine

    2016-01-01

    We designed multimodal tumor vaccine that consists of irradiated tumor cells infected with the oncolytic IL-12-expressing HSV-1 virus, M002. This vaccine was tested against the syngeneic neuroblastoma mouse model Neuro 2a injected into the right caudate nucleus of the immunocompetent A/J mice. Mice were vaccinated via intramuscular injection of multimodal vaccine or uninfected irradiated tumor cells at seven and 14 days after tumor establishment. While there was no survival difference between groups vaccinated with cell-based vaccine applied following tumor injection, a premunition prime/boost vaccination strategy produced a significant survival advantage in both groups and sustained immune response to an intracranial rechallenge of the same tumor. The syngeneic but unrelated H6 hepatocellular tumor cell line grew unrestricted in vaccinated mice, indicative of vaccine-mediated specific immunity to Neuro 2a tumors. Longitudinal analyses of tumor-infiltrating lymphocytes revealed a primary adaptive T cell response involving both CD4+ and CD8+ T cell subsets. Spleen cell mononuclear preparations from vaccinated mice were significantly more cytotoxic to Neuro 2a tumor cells than spleen cells from control mice as demonstrated in a four-hour in vitro cytotoxicity assay. These results strongly suggest that an irradiated whole cell tumor vaccine incorporating IL-12-expressing M002 HSV can produce a durable, specific immunization in a murine model of intracranial tumor. PMID:27610392

  18. Effect of HSV-IL12 Loaded Tumor Cell-Based Vaccination in a Mouse Model of High-Grade Neuroblastoma.

    PubMed

    Bauer, David F; Pereboeva, Larisa; Gillespie, G Yancey; Cloud, Gretchen A; Elzafarany, Osama; Langford, Catherine; Markert, James M; Lamb, Lawrence S

    2016-01-01

    We designed multimodal tumor vaccine that consists of irradiated tumor cells infected with the oncolytic IL-12-expressing HSV-1 virus, M002. This vaccine was tested against the syngeneic neuroblastoma mouse model Neuro 2a injected into the right caudate nucleus of the immunocompetent A/J mice. Mice were vaccinated via intramuscular injection of multimodal vaccine or uninfected irradiated tumor cells at seven and 14 days after tumor establishment. While there was no survival difference between groups vaccinated with cell-based vaccine applied following tumor injection, a premunition prime/boost vaccination strategy produced a significant survival advantage in both groups and sustained immune response to an intracranial rechallenge of the same tumor. The syngeneic but unrelated H6 hepatocellular tumor cell line grew unrestricted in vaccinated mice, indicative of vaccine-mediated specific immunity to Neuro 2a tumors. Longitudinal analyses of tumor-infiltrating lymphocytes revealed a primary adaptive T cell response involving both CD4+ and CD8+ T cell subsets. Spleen cell mononuclear preparations from vaccinated mice were significantly more cytotoxic to Neuro 2a tumor cells than spleen cells from control mice as demonstrated in a four-hour in vitro cytotoxicity assay. These results strongly suggest that an irradiated whole cell tumor vaccine incorporating IL-12-expressing M002 HSV can produce a durable, specific immunization in a murine model of intracranial tumor.

  19. [Effect of Flor-Essence on serum levels of IL-6, IL-12, TNF-α and NK cells in exercise rats].

    PubMed

    Lin, Hua; He, Ye-Heng; Xu, Rui; Zou, Wei

    2015-12-25

    The present study was aimed to investigate the effects of exercise and nutrition intervention on rat immune function. Flor-Essence is a kind of health food produced by FLORA company in Canada and certified by Quality Assurance International (QAI). Its main components are burdock root, cress leaves of grass, kelp, Turkish rhubarb root, et al. Flor-Essence has been shown to activate the body detoxification path, improve the physical environment, and inhibit cancer cell growth and proliferation. Sixty male Sprague-Dawley (SD) rats were randomly divided into six groups: control, NS + training, low-dose Flor-Essence + training, low-dose Flor-Essence, high-dose Flor-Essence + training, high-dose Flor-Essence groups. The rats in NS + training, low-dose Flor-Essence + training, high-dose Flor-Essence + training groups swam 35 min per day in the water tank for 6 days a week. One hour before exercise, the rats were given low- (2.5 mg/mL) or high-dose (5 mg/mL) Flor-Essence daily by intragastric administration, and the rats in NS + training group were given equivalent volume of NS. On the last day of four training weeks, all rats took part in a bout of exhaustive exercise, and then were sacrificed immediately. Arterial blood serum samples were taken for the assays of IL-6, IL-12 and TNF-α contents, spleens for natural killer (NK) cells activity. The results showed that serum IL-6 content in NS + training group was decreased compared with that in control group. Low- and high-dose Flor-Essence groups showed decreased IL-6, IL-12 and TNF-α serum contents, as well as longer exhaustive time, compared with control group. The improving effects of high-dose Flor- Essence on IL-6, TNF-α and exhaustive time were greater than those of low dose. Compared with NS + training, low- and high-dose Flor-Essence + training reduced serum contents of IL-6 and TNF-α, and prolonged exhaustive time; only high-dose Flor-Essence + training decreased serum IL-12 content and enhanced NK cells

  20. Two Susceptibility Loci to Takayasu Arteritis Reveal a Synergistic Role of the IL12B and HLA-B Regions in a Japanese Population

    PubMed Central

    Terao, Chikashi; Yoshifuji, Hajime; Kimura, Akinori; Matsumura, Takayoshi; Ohmura, Koichiro; Takahashi, Meiko; Shimizu, Masakazu; Kawaguchi, Takahisa; Chen, Zhiyong; Naruse, Taeko K.; Sato-Otsubo, Aiko; Ebana, Yusuke; Maejima, Yasuhiro; Kinoshita, Hideyuki; Murakami, Kosaku; Kawabata, Daisuke; Wada, Yoko; Narita, Ichiei; Tazaki, Junichi; Kawaguchi, Yasushi; Yamanaka, Hisashi; Yurugi, Kimiko; Miura, Yasuo; Maekawa, Taira; Ogawa, Seishi; Komuro, Issei; Nagai, Ryozo; Yamada, Ryo; Tabara, Yasuharu; Isobe, Mitsuaki; Mimori, Tsuneyo; Matsuda, Fumihiko

    2013-01-01

    Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B∗52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10−13, OR = 1.75, 95% CI 1.42–2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10−7, OR = 1.50, 95% CI 1.28–1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B∗52:01, overall p = 2.8 × 10−21, OR = 2.44, 95% CI 2.03–2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B∗52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor. PMID:23830516

  1. The effect of IL-2, IL-12 and IL15 on the function of natural killer cells in children suffering from type 1 diabetes mellitus.

    PubMed

    Chrul, Sławomir; Polakowska, Ewa; Mycko, Marcin; Fendler, Wojciech; Zwiech, Rafał; Szadkowska, Agnieszka

    2013-01-01

    The immunologic reaction of pancreatic islets destruction leads to the occurrence of type 1 diabetes mellitus (T1D). The autoreactive lymphocytes play the pivotal role in this process although mechanisms regulating the lymphocyte migration and infiltration of Langerhans islets have not been fully understood yet. The in vitro studies showed natural killer (NK) cells potency to initiate pancreatic islets cell lyses. Many authors postulate that NK cells may be involved in this reaction. The aim of the study was to evaluate the effect of IL-2, IL-12 and IL-15 stimulation on peripheral blood NK cells in children suffering from type 1 diabetes mellitus in comparison to healthy controls. Fifteen children with type 1 diabetes and 10 healthy adults were examined. NK cells were isolated by the magnetic cell separation system (MACS). For activation, NK cells were cultured with IL-2, IL-12 and IL-15 for 24 hours. The production of IFN-γ and IL-10 by NK cells was measured using commercial ELISA kits. FACS analysis of cell surface antigens--CD16, CD56, NKG2D and CD137 was performed using LSR II flow cytometer. In children with T1D the IFN-γ median concentration in supernatant obtained from NK cells culture was 16.831 ng/ml (inter quartile range 5.566-25.509) and did not statistically differ from median IFN-γ concentration in the control group--14.810 ng/ml (7.022-18.785), p = 0.76. In contrast, the IL-10 median concentration was statistically higher in T1D patients 7.87 pg/ml (1.32-11.37) than in healthy participants--1.41 pg/ml (1.05-4.81), p = 0.01. The median (inter-quartile range) percentage of NK NKG2D(+) was found in 0.42% (0.28-0.76) cells of TID patients versus 0.72% (0.53-1.08) in the controls (p = 0.05). There was no difference between -T1D group and the control group in regard to NK cells expressing CD137 - 6.58% (3.38-12.4) versus 6.85% (2.94-10.8); p = 0.8. The observed activity of NK cells after in vitro stimulation by IL2, IL-12 and IL15 in children suffering

  2. Skewed Helper T-Cell Responses to IL-12 Family Cytokines Produced by Antigen-Presenting Cells and the Genetic Background in Behcet's Disease

    PubMed Central

    Shimizu, Jun; Kaneko, Fumio; Suzuki, Noboru

    2013-01-01

    Behcet's disease (BD) is a multisystemic inflammatory disease and is characterized by recurrent attacks on eyes, brain, skin, and gut. There is evidence that skewed T-cell responses contributed to its pathophysiology in patients with BD. Recently, we found that Th17 cells, a new helper T (Th) cell subset, were increased in patients with BD, and both Th type 1 (Th1) and Th17 cell differentiation signaling pathways were overactivated. Several researches revealed that genetic polymorphisms in Th1/Th17 cell differentiation signaling pathways were associated with the onset of BD. Here, we summarize current findings on the Th cell subsets, their contribution to the pathogenesis of BD and the genetic backgrounds, especially in view of IL-12 family cytokine production and pattern recognition receptors of macrophages/monocytes. PMID:24490076

  3. Anticancer Cytokines: Biology and Clinical Effects of Interferon-α2, Interleukin (IL)-2, IL-15, IL-21, and IL-12.

    PubMed

    Floros, Theofanis; Tarhini, Ahmad A

    2015-08-01

    Efforts over nearly four decades have focused on ways to use cytokines to manipulate the host immune response towards cancer cell recognition and eradication. Significant advances were achieved with interleukin-2 (IL-2) and interferon-α (IFN-α), primarily in the treatment of patients with melanoma and renal cell carcinoma. However, the utility of other cytokines showing promise in the preclinical setting has not been established largely because of toxicity, the complex functionality of each cytokine and the difficulty mimicking in preclinical models the human environment. Here, we review the basic biology and the clinical experiences with IFN-α, IL-2, IL-15, IL-21, and IL-12. We will also review ongoing clinical trials and discuss future directions including potential use of cytokines in combination with other effective immunotherapy approaches that have come of age in recent years. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. D-Alanylation of Teichoic Acids and Loss of Poly-N-Acetyl Glucosamine in Staphylococcus aureus during Exponential Growth Phase Enhance IL-12 Production in Murine Dendritic Cells.

    PubMed

    Lund, Lisbeth Drozd; Ingmer, Hanne; Frøkiær, Hanne

    2016-01-01

    Staphylococcus aureus is a major human pathogen that has evolved very efficient immune evading strategies leading to persistent colonization. During different stages of growth, S. aureus express various surface molecules, which may affect the immune stimulating properties, but very little is known about their role in immune stimulation and evasion. Depending on the growth phase, S. aureus may affect antigen presenting cells differently. Here, the impact of growth phases and the surface molecules lipoteichoic acid, peptidoglycan and poly-N-acetyl glucosamine on the induction of IL-12 imperative for an efficient clearance of S. aureus was studied in dendritic cells (DCs). Exponential phase (EP) S. aureus was superior to stationary phase (SP) bacteria in induction of IL-12, which required actin-mediated endocytosis and endosomal acidification. Moreover, addition of staphylococcal cell wall derived peptidoglycan to EP S. aureus stimulated cells increased bacterial uptake but abrogated IL-12 induction, while addition of lipoteichoic acid increased IL-12 production but had no effect on the bacterial uptake. Depletion of the capability to produce poly-N-acetyl glucosamine increased the IL-12 inducing activity of EP bacteria. Furthermore, the mutant dltA unable to produce D-alanylated teichoic acids failed to induce IL-12 but like peptidoglycan and the toll-like receptor (TLR) ligands LPS and Pam3CSK4 the mutant stimulated increased macropinocytosis. In conclusion, the IL-12 response by DCs against S. aureus is highly growth phase dependent, relies on cell wall D-alanylation, endocytosis and subsequent endosomal degradation, and is abrogated by receptor induced macropinocytosis.

  5. LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing β-catenin activation in dendritic cells.

    PubMed

    Orr, Selinda J; Burg, Ashley R; Chan, Tim; Quigley, Laura; Jones, Gareth W; Ford, Jill W; Hodge, Deborah; Razzook, Catherine; Sarhan, Joseph; Jones, Yava L; Whittaker, Gillian C; Boelte, Kimberly C; Lyakh, Lyudmila; Cardone, Marco; O'Connor, Geraldine M; Tan, Cuiyan; Li, Hongchuan; Anderson, Stephen K; Jones, Simon A; Zhang, Weiguo; Taylor, Philip R; Trinchieri, Giorgio; McVicar, Daniel W

    2013-05-01

    Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

  6. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    PubMed Central

    Weinhold, Mario; Eisenblätter, Martin; Jasny, Edith; Fehlings, Michael; Finke, Antje; Gayum, Hermine; Rüschendorf, Ursula; Renner Viveros, Pablo; Moos, Verena; Allers, Kristina; Schneider, Thomas; Schaible, Ulrich E.; Schumann, Ralf R.; Mielke, Martin E.; Ignatius, Ralf

    2013-01-01

    Background Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4+ and CD8+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle. Methodology/Principal findings We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2. Conclusions/Significance Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines. PMID:23805193

  7. TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

    PubMed

    Lyakh, Lyudmila A; Sanford, Michael; Chekol, Sebel; Young, Howard A; Roberts, Anita B

    2005-02-15

    We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.

  8. Maturation of monocyte derived dendritic cells with OK432 boosts IL-12p70 secretion and conveys strong T-cell responses

    PubMed Central

    2011-01-01

    Background Design of tumour specific immunotherapies using the patients' own dendritic cells (DC) is a fast advancing scientific field. The functional qualities of the DC generated in vitro are critical, and today's gold standard for maturation is a cytokine cocktail consisting of IL-1β, IL-6, TNF-α and PGE2 generating cells lacking IL-12p70 production. OK432 is an immunotherapeutic agent derived from killed Streptococcus pyogenes that has been used clinically to treat malignant and benign neoplasms for decades. Methods In this study, we analysed the effects of OK432 on DC maturation, DC migration, cytokine and chemokine secretion as well as T-cell stimulatory capacity, and compared it to the cytokine cocktail alone and combinations of OK432 with the cytokine cocktail. Results OK432 induced a marked up-regulation of CD40 on the cell surface as well as a strong inflammatory response from the DC with significantly more secretion of 19 different cytokines and chemokines compared to the cytokine cocktail. Interestingly, secretion of IL-15 and IL-12p70 was detected at high concentrations after maturation of DC with OK432. However, the OK432 treated DC did not migrate as well as DC treated with cytokine cocktail in a transwell migration assay. During allogeneic T-cell stimulation OK432 treated DC induced proliferation of over 50 percent of CD4 and 30 percent of CD8 T-cells for more than two cell divisions, whereas cytokine cocktail treated DC induced proliferation of 12 and 11 percent of CD4 and CD8 T-cells, respectively. Conclusions The clinically approved compound OK432 has interesting properties that warrants its use in DC immunotherapy and should be considered as a potential immunomodulating agent in cancer immunotherapy. PMID:21208424

  9. IFNγ and IL-12 Restrict Th2 Responses during Helminth/Plasmodium Co-Infection and Promote IFNγ from Th2 Cells

    PubMed Central

    Coomes, Stephanie M.; Pelly, Victoria S.; Kannan, Yashaswini; Okoye, Isobel S.; Czieso, Stephanie; Entwistle, Lewis J.; Perez-Lloret, Jimena; Nikolov, Nikolay; Potocnik, Alexandre J.; Biró, Judit; Langhorne, Jean; Wilson, Mark S.

    2015-01-01

    Parasitic helminths establish chronic infections in mammalian hosts. Helminth/Plasmodium co-infections occur frequently in endemic areas. However, it is unclear whether Plasmodium infections compromise anti-helminth immunity, contributing to the chronicity of infection. Immunity to Plasmodium or helminths requires divergent CD4+ T cell-driven responses, dominated by IFNγ or IL-4, respectively. Recent literature has indicated that Th cells, including Th2 cells, have phenotypic plasticity with the ability to produce non-lineage associated cytokines. Whether such plasticity occurs during co-infection is unclear. In this study, we observed reduced anti-helminth Th2 cell responses and compromised anti-helminth immunity during Heligmosomoides polygyrus and Plasmodium chabaudi co-infection. Using newly established triple cytokine reporter mice (Il4gfpIfngyfpIl17aFP635), we demonstrated that Il4gfp+ Th2 cells purified from in vitro cultures or isolated ex vivo from helminth-infected mice up-regulated IFNγ following adoptive transfer into Rag1–/– mice infected with P. chabaudi. Functionally, Th2 cells that up-regulated IFNγ were transcriptionally re-wired and protected recipient mice from high parasitemia. Mechanistically, TCR stimulation and responsiveness to IL-12 and IFNγ, but not type I IFN, was required for optimal IFNγ production by Th2 cells. Finally, blockade of IL-12 and IFNγ during co-infection partially preserved anti-helminth Th2 responses. In summary, this study demonstrates that Th2 cells retain substantial plasticity with the ability to produce IFNγ during Plasmodium infection. Consequently, co-infection with Plasmodium spp. may contribute to the chronicity of helminth infection by reducing anti-helminth Th2 cells and converting them into IFNγ-secreting cells. PMID:26147567

  10. LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells

    PubMed Central

    Orr, Selinda J.; Burg, Ashley R.; Chan, Tim; Quigley, Laura; Jones, Gareth W.; Ford, Jill W.; Hodge, Deborah; Razzook, Catherine; Sarhan, Joseph; Jones, Yava L.; Whittaker, Gillian C.; Boelte, Kimberly C.; Lyakh, Lyudmila; Cardone, Marco; O'Connor, Geraldine M.; Tan, Cuiyan; Li, Hongchuan; Anderson, Stephen K.; Jones, Simon A.; Zhang, Weiguo; Taylor, Philip R.; Trinchieri, Giorgio; McVicar, Daniel W.

    2013-01-01

    Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2 −/− mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2−/− DCs. Accordingly, Lat2−/− DCs directed reduced Th1 polarization in vitro and Lat2 −/− mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance. PMID:23675302

  11. Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    PubMed Central

    Choi, In-Wook; Ismail, Hassan Ahmed Hassan Ahmed; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Yuk, Jae-Min; Jo, Eun-Kyeong; Lee, Young-Ha

    2015-01-01

    Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings

  12. [Effects of atovaquone and astragalus combination on the treatment and IL-2, IL-12, IFN-γ levels on mouse models of acute toxoplasmosis].

    PubMed

    Sönmez, Neşe; Büyükbaba Boral, Ozden; Kaşali, Kamber; Tekeli, Fatma

    2014-10-01

    Reactivation of Toxoplasma gondii infections and serious clinical manifestations such as encephalitis may develop in immunocompromised subjects and AIDS patients. Different protocols are used for the treatment of toxoplasmosis in high-risk patient groups, however life-long prophylactic therapy against reactivation risk in AIDS patients may lead to several undesired results. Atovaquone is an effective antiprotozoal agent against toxoplasmosis with minor side effects. On the other hand, Astragalus membranaceus root extract (AmE) has been shown to have immunomodulatory and antimicrobial activities, empowering immunity by enhancing proliferation and activation of phagocytic cells mainly macrophages, and inducing Th1 type immune response. The aim of this study was to investigate the effectiveness of atovaquone alone and in combination with AmE, in the treatment of toxoplasmosis, and on the levels of IL-2, IL-12 and IFN-γ in experimentally infected mice with T.gondii. For this purpose, four experimental groups, each consisting of eight BALB/c mice, were set with the approval of Ethics Committee for the Animal Experiments. All the mice were infected with 0.5 ml of a suspension containing 2 x 104/ml trophozoites prepared from T.gondii RH strain by intraperitoneal injection. Twenty-four hours after the infection, atovaquone (100 mg/kg/day) was given to atovaquone group, AmE (0.075 mg/g) to astragalus group and atovaquone (100 mg/kg/day) plus AmE (0.075 mg/g) to Atovaquone + Astragalus (Ato + Astra) group by oral gavage. The mice in the fourth group, which was the control group, were all infected but untreated. The above administrations were carried out for seven days. On the 8th day peritoneal fluids of mice were collected under anaesthesia and trophozoite numbers per 1 ml were detected by counting on the Thoma slide. In addition, the heart bloods of mice were drawn and IL-2, IL-12, IFN-γ levels were determined in serum samples by using commercial ELISA kits (e

  13. [Investigation of combined effectiveness of spiramycin and beta-glucan in mice models of acute toxoplasmosis and determination of IL-10, IL-12 and TNF-α levels].

    PubMed

    Büyükbaba Boral, Ozden; Sönmez Tamer, Gülden; Keçeli Özcan, Sema; Sönmez, Neşe; Işsever, Halim; Tekeli, Fatma

    2012-07-01

    Toxoplasmosis which is caused by Toxoplasma gondii, has a high risk of fetal infection development if the infection occurs during pregnancy. Treatment with oral spiramycin is recommended during pregnancy in order to prevent the transmission of protozoa to fetus and development of infection. Since beta- glucan is known to stimulate the immune system and increase the phagocytic activity of the cells, it has been shown to exhibit immunomodulatory effect on many infectious diseases. The objectives of this study were to investigate the effectiveness of beta-glucan alone and in combination with spiramycin and to determinate the levels of interlökin (IL)-10, IL-12 and tumor nekrosis factor (TNF)-α in mice experimentally infected with T.gondii. For this purpose, four experimental groups each consisting of eight BALB/c mice, were formed with the approval of Ethics Committee for the Animal Experiments. All the mice were infected with 2 ml of suspension containing 2 x 102/ml of trophozoite prepared from T.gondii RH strain (Refik Saydam National Public Health Agency, Parasitology Laboratory of Communicable Diseases Research Department, Ankara, Turkey), by intraperitoneal injection. Twenty-four hours after the infection, beta-glucan (3 mg/day) was given to the beta-glucan group, spiramycin (200 mg/kg/day) to the spiramycin group, beta-glucan (3 mg/day) plus spiramycin (200 mg/kg/day) to the beta-glucan-spiramycin (BG-S) group by oral gavage. The fourth group which was the control group was infected but untreated. The above administration was carried out for seven days. On the 8th day, under anaesthesia, 1 ml normal saline was given into the peritoneum, drawn back later and the number of trophozoites in 1 ml of peritoneal fluid was determined by counting them on the Thoma slide. Moreover, by drawing the heart blood; IL-10, IL-12, TNF-α levels were determined in serum samples by ELISA method (eBioscience Platinum, Austria). The number of trophozoites in the BG-S group was

  14. Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

    PubMed Central

    Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

    2013-01-01

    Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

  15. Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii.

    PubMed

    Ge, Yiyue; Chen, Jinling; Qiu, Xiaoyan; Zhang, Jie; Cui, Lunbiao; Qi, Yuhua; Liu, Xinjian; Qiu, Jingfan; Shi, Zhiyang; Lun, Zhaorong; Shen, Jilong; Wang, Yong

    2014-06-01

    Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88(-/-) mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88(-/-) mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m(-/-)) mice injected i.v. with MyD88(-/-) natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.

  16. IL-12 and GM-CSF in DNA/MVA Immunizations against HIV-1 CRF12_BF Nef Induced T-Cell Responses With an Enhanced Magnitude, Breadth and Quality

    PubMed Central

    Rodríguez, Ana María; Falivene, Juliana; Holgado, María Pía; Turk, Gabriela; Gherardi, María Magdalena

    2012-01-01

    In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide. This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality

  17. Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.

    PubMed

    Mukherjee, Sandip; Mukherjee, Budhaditya; Mukhopadhyay, Rupkatha; Naskar, Kshudiram; Sundar, Shyam; Dujardin, Jean-Claude; Roy, Syamal

    2014-10-15

    The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.

  18. Genetic variations in interleukin-12 related genes in immune-mediated diseases.

    PubMed

    van Wanrooij, R L J; Zwiers, A; Kraal, G; Bouma, G

    2012-12-01

    The interleukin-12 (IL-12) family comprises a group of heterodimeric cytokines and their respective receptors that play key roles in immune responses. A growing number of autoimmune diseases has been found to be associated with genetic variation in these genes. Based on their respective associations with the IL-12 genes, autoimmune diseases appear to cluster in two groups that either show strong associations with the Th1/Th17 pathway (as indicated by genetic association with IL12B and IL23R) or the Th1/IL-35 pathway as the consequence of their association with polymorphisms in the IL12A gene region. The genetic associations are described in relation to what is known of the functionality of these genes in the various diseases. Comparing association data for gene families in different diseases may lead to better insight in the function of the genes in the onset and course of the disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Light-Emitting Diode at 460 ± 20 nm Increases the Production of IL-12 and IL-6 in Murine Dendritic Cells.

    PubMed

    Carvalho-Costa, Tamires Marielem; Mendes, Maria Tays; da Silva, Marcos Vinicius; Rodrigues, Virmondes; Bruschi Thedei, Giuliana Cristina Marre; Oliveira, Carlo Jose Freire; Thedei, Geraldo

    2017-10-01

    Light emitting diode (LED) therapy has been proposed as an option for the treatment of many skin inflammatory processes. Dendritic cells (DCs) are important cells of skin that participate in the initiation and activation of skin immunity. The modulation of these cells by LED could explain much of its effects. Thus, the aim of this study was to examine the effects of LED at 460 ± 20 nm on cytokine production and the expression of surface markers on DCs. DCs were obtained from mouse bone marrow-derived dendritic cells (BMDCs). The LED was applied giving a fluence of 3.3, 8.2, or 16.5 J/cm(2) on BMDCs or lipopolysaccharide (LPS)-matured BMDCs. The production of cytokine was analyzed by enzyme linked immunosorbant assay (ELISA) and the expression of DC co- and stimulatory was analyzed markers by cytometry. LED increases IL-12p40 and IL-6 production in both nonstimulated BMDCs and LPS-matured BMDCs. The expression of MHC-II molecule was inhibited and the expression of the CD86 molecule was increased in nonstimulated BMDCs but not in LPS-matured BMDCs. The production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) and the expression of CD40 were not altered. These results demonstrate that LED stimulated cytokine production in BMDCs, suggesting a proinflammatory role in the tested conditions and maybe it can increase DC maturation.

  20. Vasoactive intestinal peptide inhibits liver pathology in acute murine schistosomiasis mansoni and modulates IL-10, IL-12 and TNF-alpha production.

    PubMed

    Allam, Gamal

    2007-01-01

    Vasoactive intestinal peptide (VIP) exerts a broad range of biologic actions that may include modulation of hepatic granuloma formation. This study aimed to investigate the effect of VIP administration on the course of acute murine schistosomiasis mansoni. Mice were infected each with 40 Schistosoma (S.) mansoni cercariae and injected intraperitoneally with VIP at a total dose of 1mug/kg body weight. VIP treatment was very effective in diminishing worm fecundity, hepatic granuloma size and number by about 54%, 75% and 51%, respectively, and reducing liver collagen content. Serum level of interleukin (IL)-10 was increased, while level of IL-12 and tumor necrosis factor (TNF)-alpha were decreased as a result of VIP administration. Carbohydrate antigen 19.9 (CA 19.9) induced by S. mansoni infection was decreased with VIP treatment. Activities of hepatic gamma-glutamyl transferase (gamma-GT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in liver tissue homogenate of infected treated mice were increased. These results indicate that suitable administration of exogenous VIP can be effective in ameliorating immunopathologic damage associated with schistosomiasis.

  1. IL12, IL10, IFNγ and TNFα Expression in Human Primary Monocytes Stimulated with Bacterial Heat Shock GroEL (Hsp64) Protein

    PubMed Central

    Nalbant, Ayten; Saygılı, Tahsin

    2016-01-01

    Actinobacillus (Aggregatibacter) actinomycetemicomitans (Aa) is a bacterium that lives in the oral cavity and plays an important role in periodontal diseases. The effect of A.actinomycetemcomitans’s heat shock family protein GroEL on host or immune cells including monocytes is quite limited. In this study, the recombinant A.actinomycetemcomitans’s GroEL protein (rAaGroEL) was used as an antigen and its effects on monocytes of peripheral blood mononuclear cells (PBMCs) was investigated. To do that, PBMCs were stimulated with rAaGroEL protein at different time points (16h to 96h) and the cytokines of CD14+ monocytes were detected with intracellular cytokine staining by Flow cytometry. Data showed that AaGroEL protein has an antigenic effect on human primary monocytes. AaGroEL protein responsive CD14 monocytes stimulates the expression of IL12, IL10, IFNγ and TNFα cytokines with different kinetics and expression profile. Therefore, A. actinomycetemcomitans’s heat shock GroEL protein can modulate innate and adaptive immune responses and contribute to an inflammatory diseases pathology. PMID:27119521

  2. Human resistin stimulates the pro-inflammatory cytokines TNF-{alpha} and IL-12 in macrophages by NF-{kappa}B-dependent pathway

    SciTech Connect

    Silswal, Nirupama; Singh, Anil K.; Aruna, Battu; Mukhopadhyay, Sangita; Ghosh, Sudip; Ehtesham, Nasreen Z. . E-mail: nas_ehtesham@yahoo.com

    2005-09-09

    Resistin, a recently discovered 92 amino acid protein involved in the development of insulin resistance, has been associated with obesity and type 2 diabetes. The elevated serum resistin in human diabetes is often associated with a pro-inflammatory milieu. However, the role of resistin in the development of inflammation is not well understood. Addition of recombinant human resistin protein (hResistin) to macrophages (both murine and human) resulted in enhanced secretion of pro-inflammatory cytokines, TNF-{alpha} and IL-12, similar to that obtained using 5 {mu}g/ml lipopolysaccharide. Both oligomeric and dimeric forms of hResistin were able to activate these cytokines suggesting that the inflammatory action of resistin is independent of its conformation. Heat denatured hResistin abrogated cytokine induction while treatment of recombinant resistin with polymyxin B agarose beads had no effect thereby ruling out the role of endotoxin in the recombinant hResistin mediated cytokine induction. The pro-inflammatory nature of hResistin was further evident from the ability of this protein to induce the nuclear translocation of NF-{kappa}B transcription factor as seen from electrophoretic mobility shift assays. Induction of TNF-{alpha} in U937 cells by hResistin was markedly reduced in the presence of either dominant negative I{kappa}B{alpha} plasmid or PDTC, a pharmacological inhibitor of NF-{kappa}B. A protein involved in conferring insulin resistance is also a pro-inflammatory molecule that has important implications.

  3. Monitoring of TNFR1, IL-2Rα, HGF, CCL8, IL-8 and IL-12p70 following HSCT and their role as GVHD biomarkers in paediatric patients.

    PubMed

    Berger, M; Signorino, E; Muraro, M; Quarello, P; Biasin, E; Nesi, F; Vassallo, E; Fagioli, F

    2013-09-01

    No predictive factors are currently available to establish patient-specific GVHD risk. A panel of six serum cytokines (TNF receptor 1, IL-2 receptor alfa (IL-2Rα), hepatocyte growth factor (HGF), monocyte chemo-attractant protein-2, IL-8, IL-12p70) were monitored at established time points (days -1, +1, +7, +14, +21, +28 and +60) in 170 paediatric hematopoietic SCT (HSCT) recipients. We found that higher concentrations of IL-2Rα on days +14 and +21 together with HGF on days +14 and +21 were significantly associated at a higher probability of both grade II-IV GVHD (on day +14 it was: 60% vs 28%, P=0.007) and grade III-IV (on day +14 it was: 40% vs 15%, P=0.001). The higher IL-8 serum concentration on day +28 was associated with a lower probability of chronic GVHD being 4% vs 29% (P=0.01) for patients with higher vs lower IL-8 serum concentration. These findings were confirmed when the analysis was restricted to the the matched unrelated donor group. In conclusion, even if the serum cytokine levels were related to several variables associated with HSCT, we identified two cytokines as predictors of GVHD II-IV and III-IV, translating into a higher TRM risk (17% vs 3%, P=0.004).

  4. Phase I Trial of Adenovirus-Mediated IL-12 Gene Transduction in Patients with Recurrent Locally Advanced Prostate Cancer Following Therapy

    DTIC Science & Technology

    2005-10-01

    radiation therapy who are presently not on hormonal therapy. An important part of the screening process is a needle biopsy of the prostate to confirm the...has been amended (see below) to also include patients who had their locally advanced prostate cancer treated with hormonal ablative therapy...the lack of effective therapies for men who have failed definitive radiotherapy or who have locally advanced cancer despite hormone ablative therapy

  5. Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.

    PubMed

    Girart, María V; Fuertes, Mercedes B; Domaica, Carolina I; Rossi, Lucas E; Zwirner, Norberto W

    2007-09-15

    NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.

  6. Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

    PubMed Central

    Holgado, María Pía; Falivene, Juliana; Gherardi, María Magdalena

    2014-01-01

    Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses. PMID:25215887

  7. SJSZ glycoprotein (38kDa) prevents thymus atrophy and enhances expression of IL-2 and IL-12 in diethylnitrosamine-induced hepatocarcinogenesis.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2012-07-01

    Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but has also been shown to provoke antitumor immune responses. Polarized T helper type 2 (Th2) responses down-regulate antitumor immunity to link with HCC. The objective of this study was to investigate the protective effect of the Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on thymus atrophy and differential response of Th1/Th2 cells induced by diethlynitrosamine (DEN). To evaluate the modulatory effect of the SJSZ glycoprotein on thymic atrophy and imbalanced Th1/Th2 cells, we examined the weight of the thymus, [(3)H]-thymidine incorporation and expression of proliferating cell nuclear antigen (PCNA), and activities of protein kinase C (PKC)/intracellular Ca(2+), extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, T-box transcription factor (T-bet), GATA-binding protein-3 (GATA-3), cytokines [interleukin (IL)-4, -10, -2, -12 and interferon (IFN)-γ] using radioactivity, immunoblot analysis, and qRT-PCR. The SJSZ glycoprotein (10mg/kg, BW) was shown to increase the weight of the thymus, [(3)H]-thymidine incorporation and PCNA in thymocytes induced by DEN. Also, it increased expression levels of T-bet and Th1 cytokines (IFN-γ, IL-2 and IL-12). However, the activity of PKC/intracellular Ca(2+), phosphorylation of ERK and p38 MAPK, expression levels of GATA-3 and Th2 cytokines (IL-4 and IL-10) were decreased. Taken together, these results suggest that the SJSZ glycoprotein can prevent thymic atrophy and Th2 cytokines induced by DEN. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection.

    PubMed

    Fernández-Santoscoy, María; Wenzel, Ulf A; Yrlid, Ulf; Cardell, Susanna; Bäckhed, Fredrik; Wick, Mary Jo

    2015-01-01

    The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4(+), and CD8(+) T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth.

  9. The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection

    PubMed Central

    Fernández-Santoscoy, María; Wenzel, Ulf A.; Yrlid, Ulf; Cardell, Susanna; Bäckhed, Fredrik; Wick, Mary Jo

    2015-01-01

    The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4+, and CD8+ T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth. PMID:26734581

  10. Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

    PubMed Central

    Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

    2013-01-01

    Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

  11. Aβ42 gene vaccine prevents Aβ42 deposition in brain of double transgenic mice

    PubMed Central

    Qu, Bao-Xi; Xiang, Qun; Li, Liping; Johnston, Stephen Albert; Hynan, Linda S.; Rosenberg, Roger N.

    2008-01-01

    Aβ42 peptide aggregation and deposition is an important component of the neuropathology of Alzheimer’s disease (AD). Gene-gun mediated gene vaccination targeting Aβ42 is a potential method to prevent and treat AD. APPswe/PS1ΔE9 transgenic (Tg) mice were immunized with an Aβ42 gene construct delivered by the gene gun. The vaccinated mice developed Th2 antibodies (IgG1) against Aβ42. The Aβ42 levels in brain were decreased by 41% and increased in plasma 43% in the vaccinated compared with control mice as assessed by ELISA analysis. Aβ42 plaque deposits in cerebral cortex and hippocampus were reduced by 51% and 52%, respectively, as shown by quantitative immunolabeling. Glial cell activation was also significantly attenuated in vaccinated compared with control mice. One rhesus monkey was vaccinated and developed anti-Aβ42 antibody. These new findings advance significantly our knowledge that gene-gun mediated Aβ42 gene immunization effectively induces a Th2 immune response and reduces the Aβ42 levels in brain in APPswe/PS1ΔE9 mice. Aβ42 gene vaccination may be safe and efficient immunotherapy for AD. PMID:17574274

  12. Associations of IL-4, IL-6, and IL-12 levels in peripheral blood with lung function, cellular immune function, and quality of life in children with moderate-to-severe asthma.

    PubMed

    Cui, Ai-Hua; Zhao, Jing; Liu, Shu-Xiang; Hao, Ying-Shuang

    2017-03-01

    Pediatric asthma has gained increasing concerns with poorly understood pathogenesis. The purpose of this study was to explore the associations of interleukin-4 (IL-4), IL-6, and IL-12 levels in peripheral blood (PB) with lung function, cellular immune function, and children's quality of life (QOL) with moderate-to-severe asthma. A total of 1158 children with moderate-to-severe asthma (the experimental group) and 1075 healthy children (the control group) were recruited for our study. Enzyme-linked immunosorbent assay was used to detect IL-4, IL-6, and IL-12 levels. T lymphocytes were detected by alkaline phosphatase antialkaline phosphatase, and erythrocyte immune was measured by red blood cell C 3b receptor (RBC-C3bR) rosette-forming test. The forced expiratory volume in 1 second (FEV1) and peak expiratory flow (PEF) were detected, after which FEV1/forced vital capacity (FVC) was calculated before and after treatment. PedsQL3.0 was used to measure the effect of asthma on QOL of children, and the correlation between IL-4, IL-6, and IL-12 levels and the lung function and QOL was measured. Logistic regression analysis was applied to detect related factors of moderate-to-severe asthma of children. After treatment, the decreased IL-4 and IL-6 levels and increased IL-12 level were revealed in the experimental group. The cellular immune function's disorder was significantly decreased, and an elevated CD3, CD4, CD8, and declined CD4/CD8 level was performed in T lymphocytes. RBC-C3bR was increased, and red blood cell immune complex (RBC-IC) was reduced in erythrocyte immune in comparison with those before treatment. Lung function parameters all increased. After treatment, the symptoms of asthma in children reduced with scores of increased QOL. IL-4 was positively related to RBC-IC, but negatively associated with the QOL score. IL-6 showed negative connection with CD4/CD8, RBC-C3bR, FEV1/FVC, and QOL score, and had positive connection with PEF. In addition, IL-12 was

  13. Associations of IL-4, IL-6, and IL-12 levels in peripheral blood with lung function, cellular immune function, and quality of life in children with moderate-to-severe asthma

    PubMed Central

    Cui, Ai-Hua; Zhao, Jing; Liu, Shu-Xiang; Hao, Ying-Shuang

    2017-01-01

    Abstract Background: Pediatric asthma has gained increasing concerns with poorly understood pathogenesis. The purpose of this study was to explore the associations of interleukin-4 (IL-4), IL-6, and IL-12 levels in peripheral blood (PB) with lung function, cellular immune function, and children's quality of life (QOL) with moderate-to-severe asthma. Methods: A total of 1158 children with moderate-to-severe asthma (the experimental group) and 1075 healthy children (the control group) were recruited for our study. Enzyme-linked immunosorbent assay was used to detect IL-4, IL-6, and IL-12 levels. T lymphocytes were detected by alkaline phosphatase antialkaline phosphatase, and erythrocyte immune was measured by red blood cell C 3b receptor (RBC-C3bR) rosette-forming test. The forced expiratory volume in 1 second (FEV1) and peak expiratory flow (PEF) were detected, after which FEV1/forced vital capacity (FVC) was calculated before and after treatment. PedsQL3.0 was used to measure the effect of asthma on QOL of children, and the correlation between IL-4, IL-6, and IL-12 levels and the lung function and QOL was measured. Logistic regression analysis was applied to detect related factors of moderate-to-severe asthma of children. Results: After treatment, the decreased IL-4 and IL-6 levels and increased IL-12 level were revealed in the experimental group. The cellular immune function's disorder was significantly decreased, and an elevated CD3, CD4, CD8, and declined CD4/CD8 level was performed in T lymphocytes. RBC-C3bR was increased, and red blood cell immune complex (RBC-IC) was reduced in erythrocyte immune in comparison with those before treatment. Lung function parameters all increased. After treatment, the symptoms of asthma in children reduced with scores of increased QOL. IL-4 was positively related to RBC-IC, but negatively associated with the QOL score. IL-6 showed negative connection with CD4/CD8, RBC-C3bR, FEV1/FVC, and QOL score, and had positive connection

  14. Expression of interferon-gamma (IFN-δ), IL-10, IL-12 and transforming growth factor-beta (TGF-β) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA)

    PubMed Central

    BUCHT, A.; LARSSON, P.; WEISBROT, L.; THORNE, C.; PISA, P.; SMEDEGÅRD, G.; KEYSTONE, E C; GRÖNBERG, A.

    1996-01-01

    The expression of immunoregulatory cytokines was investigated in freshly isolated synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with RA, using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. IFN-γ, TGF-β, IL-10 and IL-12 (p40) transcripts were detected in SFMC of patients with early disease (<1 year duration) as well as in patients with long standing arthritis (>1 year). The expression of IFN-γ, IL-10 and IL-12 mRNA was increased in SFMC compared with RA PBMC. In addition, the expression was higher in RA SFMC than in PBMC from healthy control individuals. Immunoassay analysis of the secreted IL-12 heterodimer demonstrated increased levels in RA SF compared with levels found in serum from RA patients and control individuals. High levels of TGF-β mRNA were found in SFMC, but a significantly decreased TGF-β/β2-microglobulin (β2-M) ratio was found compared with PBMC from both patients and control individuals. IL-4 mRNA could not be detected, either in SFMC or in PBMC. Cytokine expression in RA PBMC did not differ from control PBMC, with the exception of a decreased TGF-β/β2-M ratio in RA patients with early disease. Our findings of IFN-7 mRNA and IL-12, but undetectable levels of IL-4 mRNA, suggest that the synovitis is characterized by a type 1 immune response. The presence of TGF-β and IL-10 mRNA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level of expression may not be sufficient for down-modulation of immune activation. PMID:8608632

  15. Mycobacterium tuberculosis promotes Th17 expansion via regulation of human dendritic cells toward a high CD14 and low IL-12p70 phenotype that reprograms upon exogenous IFN-γ.

    PubMed

    Søndergaard, Jonas Nørskov; Laursen, Janne Marie; Rosholm, Lisbeth Buus; Brix, Susanne

    2014-12-01

    The capacity to develop protective immunity against mycobacteria is heterogeneously distributed among human beings, and it is currently unknown why the initial immune response induced against Mycobacterium tuberculosis (Mtb) does not provide proper clearance of this pathogen. Dendritic cells (DCs) are some of the first cells to interact with Mtb and they play an essential role in development of protective immunity against Mtb. Given that Mtb-infected macrophages have difficulties in degrading Mtb, they need help from IFN-γ-producing CD4+ T cells propagated via IL-12p70-producing DCs. Here we report that Mtb modifies human DC plasticity by expanding a CD14+ DC subset with weak IL-12p70-producing capacity. The CD14+ Mtb-promoted subset was furthermore poor inducers of IFN-γ by naive CD4+ T cells, but instead prompted IL-17A-producing RORγT+ CD4+ T cells. Mtb-derived peptidoglycan and mannosylated lipoarabinomannan partly recapitulated the subset partition induced by Mtb. Addition of IFN-γ, but neither IL-17A nor IL-22, which are potentially produced by Mtb-exposed γ/δ-T cells in mucosal linings, inhibited the differentiation toward CD14+ DCs and promoted high-level IL-12p70 in Mtb-challenged DCs. We conclude that Mtb exploits DC plasticity to reduce production of IL-12p70, and that this process is entirely divertible by exogenous IFN-γ. These data suggest that strategies to increase local IFN-γ production in the lungs of tuberculosis patients may boost host immunity toward Mtb.

  16. Phenotypic and functional activation of hyporesponsive KIRnegNKG2Aneg human NK-cell precursors requires IL12p70 provided by Poly(I:C)-matured monocyte-derived dendritic cells.

    PubMed

    Curran, Shane A; Romano, Emanuela; Kennedy, Michael G; Hsu, Katharine C; Young, James W

    2014-10-01

    A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFNγ was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFNγ production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism.

  17. Association Analysis of IL10, TNF-α, and IL23R-IL12RB2 SNPs with Behçet’s Disease Risk in Western Algeria

    PubMed Central

    Khaib Dit Naib, Ouahiba; Aribi, Mourad; Idder, Aicha; Chiali, Amel; Sairi, Hakim; Touitou, Isabelle; Lefranc, Gérard; Barat-Houari, Mouna

    2013-01-01

    Objective: We have conducted the first study of the association of interleukin (IL)-10, tumor necrosis factor alpha (TNF-α), and IL23R-IL12RB2 region single nucleotide polymorphisms (SNPs) with Behçet’s disease (BD) in Western Algeria. Methods: A total of 51 BD patients and 96 unrelated controls from West region of Algeria were genotyped by direct sequencing for 11 SNPs including 2 SNPs from the IL10 promoter [c.-819T > C (rs1800871), c.-592A > C (rs1800872)], 6 SNPs from the TNF-α promoter [c.-1211T > C (rs1799964), c.-1043C > A (rs1800630), c.-1037C > T (rs1799724), c.-556G > A (rs1800750), c.-488G > A (rs1800629), and c.-418G > A (rs361525)], and 3 SNPs from the IL23R-IL12RB2 region [g.67747415A > C (rs12119179), g.67740092G > A (rs11209032), and g.67760140T > C (rs924080)]. Results: The minor alleles c.-819T and c.-592A were significantly associated with BD [odds ratio (OR) = 2.18; 95% confidence interval (CI) 1.28–3.73, p = 0.003]; whereas, there was weaker association between TNF-α promoter SNPs or IL23R-IL12RB2 region and disease risk. Conclusion: Unlike the TNF-α and the IL23R-IL12RB2 region SNPs, the two IL10 SNPs were strongly associated with BD. The -819T, and -592A alleles and the -819TT, -819CT, and -592AA and -592CA genotypes seem to be highly involved in the risk of developing of BD in the population of Western Algeria. PMID:24151497

  18. Endomorphins 1 and 2 inhibit IL-10 and IL-12 production and innate immune functions, and potentiate NF-kappaB DNA binding in THP-1 differentiated to macrophage-like cells.

    PubMed

    Azuma, Y; Ohura, K

    2002-09-01

    We evaluated immunological effects of opioid peptides endomorphins 1 and 2 on the production of interleukin-10 (IL-10) and IL-12 cytokines, functions related to innate immunity and NF-kappaB DNA binding in human cell line THP-1. Endomorphins 1 and 2 inhibited lipopolysaccharide (LPS)-stimulated IL-10 and IL-12 production in THP-1 differentiated to macrophage-like cells by phorbol 12-myristate 13-acetate (PMA). Similarly, they suppressed LPS-stimulated IL-10 and IL-12 production in THP-1 matured to monocytes by 1alpha,25-dihydroxyvitamin D3. In addition, endomorphins 1 and 2 led to marked potentiation of NF-kappaB binding in THP-1 differentiated to macrophage-like cells. Furthermore, these endomorphins further potentiated LPS-induced NF-kappaB binding. Moreover, they inhibited chemotaxis, phagocytosis of Escherichia coli and PMA-stimulated production of hydrogen peroxide in THP-1 differentiated to macrophage-like cells. These results suggest that endomorphins 1 and 2 may inhibit THP-1 functions, such as cytokine production and functions related to innate immune, and potentiate NF-kappaB DNA binding in THP-1.

  19. Combination of interleukin-12 gene therapy, metronomic cyclophosphamide and DNA cancer vaccination directs all arms of the immune system towards tumor eradication.

    PubMed

    Denies, Sofie; Cicchelero, Laetitia; Van Audenhove, Isabel; Sanders, Niek N

    2014-08-10

    In this work a combination therapy that acts upon the immune suppressive, the innate and specific arms of the immune system is proposed. This combination therapy, which consists of intratumoral interleukin-12 (IL-12) gene therapy, human tyrosinase (hTyr) DNA vaccination and metronomic cyclophosphamide (CPX), was evaluated in a B16-F10 mouse model. The following groups were compared: (1) no treatment, (2) control vector, (3) intratumoral IL-12 gene therapy, (4) intratumoral IL-12 gene therapy+metronomic CPX, (5) intratumoral IL-12 gene therapy+metronomic CPX+hTyr DNA vaccination. Next to clinical efficacy and safety, we characterized acute effects of IL-12 and anti-tumor immune response after a second tumor challenge. All treatment groups showed increased survival and higher cure rates than control groups. Survival of non-cured mice was increased when metronomic CPX was combined with IL-12 gene therapy. Furthermore, mice that received metronomic CPX had significantly lower percentages of regulatory T cells. Addition of the hTyr DNA vaccine increased cure rate and resulted in increased survival compared to other treatment groups. We also demonstrated that the manifest necrosis within days after IL-12 gene therapy is at least partly due to IL-12 mediated activation of NK cells. All cured mice were resistant to a second challenge. A humoral memory response against the tumor cells was observed in all groups that received IL-12 gene therapy, while a cellular memory response was observed only in the vaccinated mice. In conclusion, every component of this combination treatment contributed a unique immunologic trait with associated clinical benefits.

  20. Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles

    PubMed Central

    1994-01-01

    We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen- specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL- 2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4

  1. Interleukin-12B gene polymorphism frequencies in Egyptians and sex-related susceptibility to hepatitis C infection.

    PubMed

    Youssef, Samar Samir; Abd El Aal, Asmaa Mostafa; Nasr, Amal Soliman; el Zanaty, Taher; Seif, Sameh Mohamed

    2013-08-01

    Hepatitis C virus (HCV) infection is a major health problem worldwide. Egypt is the country with the highest HCV infection epidemic in the world. Interleukin (IL)-12 is a cytokine that has been shown to have a potent role as an antiviral cytokine. IL-12 is a heterodimer of the polypeptides p35 and p40. IL-12 B, the gene encoding IL-12 p40, is polymorphic, and a functional single-nucleotide polymorphism (SNP) of the 3'-untranslated region at position rs3212227 was associated with apparent resistance to HCV. The genotype distribution of this polymorphism differs by race. This study is sought to identify the genotype distribution of the IL-12 SNP rs3212227 polymorphism in Egyptians and to assess its role in susceptibility to chronic HCV infection alone or in a sex-dependent way. The study included 238 subjects: 100 healthy controls and 138 patients with HCV infection. The IL-12 SNP rs3212227 was genotyped by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Results showed a genotype frequency of 46%, 39%, and 15% for AA, AC, and CC IL-12 genotypes, respectively. No significant result (P=0.5) was shown in the differential distribution of the IL-12 SNP genotypes between controls and patients with HCV infection. Nonetheless, this difference in the IL-12 genotype distribution was significant (0.005) when it was stratified according to sex; moreover, the C allele distribution in men and women differed with a statistically high significance (P=0.0001) in controls versus HCV patients. In conclusion, the IL-12 SNP rs3212227 polymorphism confers a susceptibility to HCV infection in a sex-dependent way in Egyptians.

  2. In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy

    PubMed Central

    ORLEANS-LINDSAY, J K; DERU, A; CRAIG, J I O; PRENTICE, H G; LOWDELL, M W

    2003-01-01

    The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-γ) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA–/CCR7– subsets known to be associated with immediate cytotoxic functions. PMID:12930376

  3. In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy.

    PubMed

    Orleans-Lindsay, J K; Deru, A; Craig, J I O; Prentice, H G; Lowdell, M W

    2003-09-01

    The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.

  4. MicroRNA-21 limits in vivo immune response-mediated activation of the IL-12/interferon gamma pathway, Th1 polarization, and the severity of delayed-type hypersensitivity1

    PubMed Central

    Lu, Thomas X.; Hartner, Jochen; Lim, Eun-Jin; Fabry, Victoria; Mingler, Melissa K.; Cole, Eric T.; Orkin, Stuart H.; Aronow, Bruce J.; Rothenberg, Marc E.

    2011-01-01

    An altered balance between Th1 and Th2 cytokines is responsible for a variety of immuno-inflammatory disorders such as asthma, yet the role of post-transcriptional mechanisms, such as those mediated by microRNAs, in adjusting the relative magnitude and balance of Th cytokine expression have been largely unexplored. Here we show that miR-21 has a central role in setting a balance between Th1 and Th2 responses to antigens. Targeted ablation of miR-21 in mice led to reduced lung eosinophilia after allergen challenge, with a broadly reprogrammed immunoactivation transcriptome, and significantly increased levels of the Th1 cytokine IFNγ. Biological network-based transcriptome analysis of OVA-challenged miR-21-/-mice identified an unexpected prominent dysregulation of IL-12/IFNγ pathways as the most significantly affected in the lungs with a key role for miR-21 in IFNγ signaling and T-cell polarization, consistent with a functional miR-21 binding site in IL-12p35. In support of these hypotheses, miR-21 deficiency led dendritic cells to produce more IL-12 after LPS stimulation, and OVA-challenged CD4+ T lymphocytes to produce increased IFNγ and decreased IL-4. Further, loss of miR-21 significantly enhanced the Th1-associated delayed-type hypersensitivity cutaneous responses. Thus, our results define miR-21 as a major regulator of Th1 vs. Th2 responses, defining a new mechanism for regulating polarized immuno-inflammatory responses. PMID:21849676

  5. The synthesis of Rantes, G-CSF, IL-4, IL-5, IL-6, IL-12 and IL-13 in human whole-blood cultures is modulated by an extract from Eleutherococcus senticosus L. roots.

    PubMed

    Schmolz, M W; Sacher, F; Aicher, B

    2001-05-01

    An ethanol extract derived from the roots of Eleutherococcus senticosus was found to influence markedly the cytokine synthesis of activated whole blood cultures of ten healthy volunteers. Whereas the synthesis of Rantes was increased over a wide range of concentrations, the release of IL-4, IL-5 and IL-12 was significantly inhibited. An inhibition at higher concentrations, switching to a stimulation at lower doses of the extract was seen with G-CSF, IL-6 and IL-13. From these particular immuno-pharmacological effects of Eleutherococcus senticosus we suggest this herbal preparation possesses immuno-modulatory potency, rather than just being immuno-suppressive or -stimulating.

  6. Gene therapy based on interleukin-12 loaded chitosan nanoparticles in a mouse model of fibrosarcoma

    PubMed Central

    Soofiyani, Saiedeh Razi; Hallaj-Nezhadi, Somayeh; Lotfipour, Farzaneh; Hosseini, Akbar Mohammad; Baradaran, Behzad

    2016-01-01

    Objective(s): Interleukin-12 (IL-12) as a cytokine has been proved to have a critical role in stimulating the immune system and has been used as immunotherapeutic agents in cancer gene therapy. Chitosan as a polymer, with high ability of binding to nucleic acids is a good candidate for gene delivery since it is biodegradable, biocompatible and non-allergenic polysaccharide. The objective of the present study was to investigate the effects of cells transfected with IL-12 loaded chitosan nanoparticles on the regression of fibrosarcoma tumor cells (WEHI-164) in vivo. Materials and Methods: WEHI-164 tumor cells were transfected with IL-12 loaded chitosan nanoparticles and then were injected subcutaneously to inoculate tumor in BALB/c mice. Tumor volumes were determined and subsequently extracted after mice sacrifice. The immunohistochemistry staining was performed for analysis of Ki-67 expression (a tumor proliferation marker) in tumor masses. The expression of IL-12 and IFN-γ were studied using real-time polymerase chain reaction and immunoblotting. Results: The group treated with IL-12 loaded chitosan nanoparticles indicated decreasing of tumor mass[r1] volume (P<0.001). The results of western blotting and real-time PCR showed that the IL-12 expression was increased in the group. Immunohistochemistry staining indicated that the Ki-67expression was reduced in the group treated with IL-12 loaded chitosan nanoparticles. Conclusion: IL-12 gene therapy using chitosan nanoparticles has therapeutic effects on the regression of tumor masses in fibrosarcoma mouse model. PMID:27917281

  7. Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy.

    PubMed

    Kamensek, Urska; Tesic, Natasa; Sersa, Gregor; Kos, Spela; Cemazar, Maja

    2017-01-01

    Electrotransfer mediated delivery of interleukin-12 (IL-12) gene, encoded on a plasmid vector, has already been demonstrated to have a potent antitumor efficacy and great potential for clinical application. In the present study, our aim was to construct an optimized IL-12-encoding plasmid that is safe from the regulatory point of view. In light of previous studies demonstrating that IL-12 should be released in a tumor localized manner for optimal efficacy, the strong ubiquitous promoter was replaced with a weak endogenous promoter of the collagen 2 gene, which is specific for fibroblasts. Next, to comply with increasing regulatory demands for clinically used plasmids, the expression cassette was cloned in a plasmid lacking the antibiotic resistance gene. The constructed fibroblast-specific and antibiotic-free IL-12 plasmid was demonstrated to support low IL-12 expression after gene electrotransfer in selected cell lines. Furthermore, the removal of antibiotic resistance did not affect the plasmid expression profile and lowered its cytotoxicity. With optimal IL-12 expression and minimal transgene non-specific effects, i.e., low cytotoxicity, the constructed plasmid could be especially valuable for different modern immunological approaches to achieve localized boosting of the host's immune system.

  8. Induction of pro-inflammatory mediators in Plasmodium berghei infected BALB/c mice breaks blood-brain-barrier and leads to cerebral malaria in an IL-12 dependent manner.

    PubMed

    Schmidt, Kim E; Schumak, Beatrix; Specht, Sabine; Dubben, Bettina; Limmer, Andreas; Hoerauf, Achim

    2011-09-01

    A severe complication of Plasmodium infection is cerebral malaria, a condition mainly attributed to overwhelming inflammatory immune reactions of the host. Murine models differing in susceptibility to experimental cerebral malaria (ECM) allow detailed studies of the host response. We show that ECM- resistant BALB/c mice were driven into interferon gamma- and IL-12-dependent ECM and subsequent death if they received CpG-oligonucleotides after Plasmodium berghei ANKA (PbA) infection. CpG application triggered production of pro-inflammatory cytokines systemically as well in spleen and brain and induced neuropathological symptoms, leading to increased mortality. Experiments with genetically deficient mice confirmed the role of IFN-γ and IL-12 during CpG-triggered immunopathology. Furthermore, the application of CpG and downstream production of pro-inflammatory cytokines contributed to the break down of the blood brain barrier visualized by Evan's Blue, comparable to PbA-infected C57BL/6 mice. Taken together, resistance of BALB/c mice towards ECM development could be altered through induction of pro-inflammatory cytokines by CpG. Therefore, approaches discussed earlier to induce pro-inflammatory immune reactions for malaria protection should be considered with caution. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  9. Activation and cytokine profile of monocyte derived dendritic cells in leprosy: in vitro stimulation by sonicated Mycobacterium leprae induces decreased level of IL-12p70 in lepromatous leprosy

    PubMed Central

    Braga, André Flores; Moretto, Daniela Ferraz; Gigliotti, Patrícia; Peruchi, Mariela; Vilani-Moreno, Fátima Regina; Campanelli, Ana Paula; Latini, Ana Carla Pereira; Iyer, Anand; Das, Pranab Kumar; de Souza, Vânia Nieto Brito

    2015-01-01

    Dendritic cells (DCs) play a pivotal role in the connection of innate and adaptive immunity of hosts to mycobacterial infection. Studies on the interaction of monocyte-derived DCs (MO-DCs) using Mycobacterium leprae in leprosy patients are rare. The present study demonstrated that the differentiation of MOs to DCs was similar in all forms of leprosy compared to normal healthy individuals. In vitro stimulation of immature MO-DCs with sonicated M. leprae induced variable degrees of DC maturation as determined by the increased expression of HLA-DR, CD40, CD80 and CD86, but not CD83, in all studied groups. The production of different cytokines by the MO-DCs appeared similar in all of the studied groups under similar conditions. However, the production of interleukin (IL)-12p70 by MO-DCs from lepromatous (LL) leprosy patients after in vitro stimulation with M. leprae was lower than tuberculoid leprosy patients and healthy individuals, even after CD40 ligation with CD40 ligand-transfected cells. The present cumulative findings suggest that the MO-DCs of LL patients are generally a weak producer of IL-12p70 despite the moderate activating properties ofM. leprae. These results may explain the poor M. leprae-specific cell-mediated immunity in the LL type of leprosy. PMID:26222022

  10. Activation and cytokine profile of monocyte derived dendritic cells in leprosy: in vitro stimulation by sonicated Mycobacterium leprae induces decreased level of IL-12p70 in lepromatous leprosy.

    PubMed

    Braga, André Flores; Moretto, Daniela Ferraz; Gigliotti, Patrícia; Peruchi, Mariela; Vilani-Moreno, Fátima Regina; Campanelli, Ana Paula; Latini, Ana Carla Pereira; Iyer, Anand; Das, Pranab Kumar; Souza, Vânia Nieto Brito de

    2015-08-01

    Dendritic cells (DCs) play a pivotal role in the connection of innate and adaptive immunity of hosts to mycobacterial infection. Studies on the interaction of monocyte-derived DCs (MO-DCs) using Mycobacterium leprae in leprosy patients are rare. The present study demonstrated that the differentiation of MOs to DCs was similar in all forms of leprosy compared to normal healthy individuals. In vitro stimulation of immature MO-DCs with sonicated M. leprae induced variable degrees of DC maturation as determined by the increased expression of HLA-DR, CD40, CD80 and CD86, but not CD83, in all studied groups. The production of different cytokines by the MO-DCs appeared similar in all of the studied groups under similar conditions. However, the production of interleukin (IL)-12p70 by MO-DCs from lepromatous (LL) leprosy patients after in vitro stimulation with M. leprae was lower than tuberculoid leprosy patients and healthy individuals, even after CD40 ligation with CD40 ligand-transfected cells. The present cumulative findings suggest that the MO-DCs of LL patients are generally a weak producer of IL-12p70 despite the moderate activating properties ofM. leprae. These results may explain the poor M. leprae-specific cell-mediated immunity in the LL type of leprosy.

  11. Anti-tumour necrosis factor (TNF) α treatment of rheumatoid arthritis (infliximab) selectively down regulates the production of interleukin (IL) 18 but not of IL12 and IL13

    PubMed Central

    Pittoni, V; Bombardieri, M; Spinelli, F; Scrivo, R; Alessandri, C; Conti, F; Spadaro, A; Valesini, G

    2002-01-01

    Methods: Ten patients with RA not responding to disease modifying antirheumatic drugs (DMARDs) received intravenous infliximab at a dose of 3 mg/kg at baseline and after two and six weeks. Serum samples were collected from all patients before each infusion and assayed for IL18, IL12, and IL13 by enzyme linked immunosorbent assay (ELISA); IL18 was also measured eight weeks after the last infusion. Results: Serum concentrations of IL18 in all patients were already markedly reduced from baseline after two weeks (p<0.005). Serum IL18 was also decreased in a stable manner after six (p<0.01) and 14 weeks (p<0.01) compared with baseline concentrations. No significant modifications were found in serum concentrations of IL12 and IL13 at any time point. Conclusion: There was a rapid and persistent decrease in serum concentrations of IL18 in all the patients studied. This result provides evidence of an in vivo regulation of IL18 by TNFα and suggests that anti-TNFα therapy is likely to interrupt the synergistic effect between these two cytokines. PMID:12117680

  12. Bone marrow-derived macrophages grown in GM-CSF or M-CSF differ in their ability to produce IL-12 and to induce IFN-gamma production after stimulation with Trypanosoma cruzi antigens.

    PubMed

    Tadokoro, C E; de Almeida Abrahamsohn, I

    2001-05-01

    Trypanosoma cruzi is the etiological agent of Chagas' disease in man. Control of parasitism at the beginning of experimental infection depends on cytokine-activated macrophages that synthesize nitric oxide (NO). We investigated macrophage populations derived in the presence of M-CSF (M-MØ) or GM-CSF (GM-MØ) regarding their ability to control intracellular parasitism by T. cruzi and to synthesize IL-12 and NO. Both macrophage populations supported intracellular multiplication of the parasite; when activated by IFN-gamma, GM-MØ exerted better control of parasitism. Stimulation of GM-MØ with T. cruzi or Staphylococcus aureus resulted in IL-12 production and higher levels of NO synthesis in comparison with stimulated M-MØ. Mice immunized with parasite-Ag-pulsed GM-MØ but not with pulsed M-MØ had increased IFN-gamma and IL-2 production in lymph nodes. However, when immunization was followed by infection with live parasites, transient elevation of IFN-gamma production was observed in both GM-MØ- and M-MØ-immunized mice, without reduction of blood parasite levels.

  13. Monocyte-derived dendritic cells from chagasic patients vs healthy donors secrete differential levels of IL-10 and IL-12 when stimulated with a protein fragment of Trypanosoma cruzi heat-shock protein-70.

    PubMed

    Cuellar, Adriana; Santander, Sandra Paola; Thomas, María Del Carmen; Guzmán, Fanny; Gómez, Alberto; López, Manuel Carlos; Puerta, Concepción J

    2008-01-01

    We analyzed the effect of the truncated heat-shock protein 70 from Trypanosoma cruzi on maturation of human dendritic cells (DCs) derived from monocytes of peripheral blood mononuclear cells from healthy donors and chagasic patients. The results show that the T-HSP70 is capable of maturing human DCs inducing an increase in the expression level of the CD83, CD86 and human leukocyte antigen-DR surface markers, as well as in the secretion of interleukin (IL)-12, tumor necrosis factor-alpha (TNF-alpha) and IL-6 cytokines. Results also show the existence of a differential functional activity of matured DCs from chagasic patients vs healthy donors in response to T-HSP70 protein and to HSP-70-derived A72 peptide, as only T-HSP70-matured DCs from chagasic patients have an enhanced secretion of IL-10 and a reduced secretion of IL-12. Moreover, the addition of A72 peptide to immature DCs from chagasic patients induced an increase in the percentage of cells expressing CD83 and CD86 molecules regarding to the expression level observed by cells from healthy donors. These findings suggest that T. cruzi HSP70 protein may induce a specific maturation profile on chagasic patients' DCs, which would favor the persistence of the parasite in the human host.

  14. Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

    PubMed Central

    Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

    2003-01-01

    AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

  15. IL-2 and IL-4 counteract budesonide inhibition of GM-CSF and IL-10, but not of IL-8, IL-12 or TNF-α production by human mononuclear blood cells

    PubMed Central

    Larsson, Susanne; Löfdahl, Claes-Göran; Linden, Margareta

    1999-01-01

    The combination of interleukin-2 (IL-2) and IL-4 reduces the inhibitory effects of glucocorticoids on granulocytemacrophage colonystimulating factor (GM-CSF) production, in agreement with the hypothesis that this combination causes glucocorticoid resistance. Whether a general cytokine resistance to glucocorticoids is induced by IL-2 and IL-4 has not been reported. Mononuclear blood cells from healthy individuals were pretreated with IL-2, IL-4, or IL-2+ IL-4 (31.3–500 U ml−1) for 48 h, prior to lipopolysaccharide (LPS; 10 ng ml−1; 20 h) and budesonide addition. Cytokine levels in the supernatants were analysed using specific immunoassays. DNA content was analysed to estimate cell numbers. GM-CSF production was totally inhibited by budesonide at 10−8 M in vehicle treated cultures, while IL-10 was inhibited to 33.4±4.3% of control. IL-2, IL-4, or IL-2+IL-4 reduced the inhibitory effects of budesonide on GM-CSF to similar levels (23.7±6.7, 31.6±8.5 and 35.1±4.3% of control, respectively). IL-2, IL-4, or IL-2+IL-4 also reduced the inhibitory effects of budesonide on IL-10 production (46.5±6.6, 55.9±7.3%, and 68.3±9.9% of control, respectively). In contrast, IL-8, IL-12 and TNF-α production did not become resistant to budesonide. Thus, glucocorticoid resistance induced by IL-2 and IL-4 is not general at the cytokine production level. While the glucocorticoid sensitivity of GM-CSF and IL-10 production decreased, the sensitivity of IL-8, IL-12 or TNF-α production was unchanged. Also, the mixture of IL-2 and IL-4 is not crucial for induction of glucocorticoid resistance of GM-CSF production. PMID:10433506

  16. Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

    PubMed

    Tanaka, Sachi; Koizumi, Shin-ichi; Masuko, Kazutaka; Makiuchi, Naoko; Aoyagi, Yuka; Quivy, Emi; Mitamura, Rieko; Kano, Tsutomu; Ohkuri, Takayuki; Wakita, Daiko; Chamoto, Kenji; Kitamura, Hidemitsu; Nishimura, Takashi

    2011-02-01

    Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism.

    PubMed

    Weiss, Gudrun; Rasmussen, Simon; Zeuthen, Louise Hjerrild; Nielsen, Birgit Nøhr; Jarmer, Hanne; Jespersen, Lene; Frøkiaer, Hanne

    2010-10-01

    Lactobacilli are probiotics that, among other health-promoting effects, have been ascribed immunostimulating and virus-preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin-12 (IL-12) -inducing properties. As IL-12 production depends on the up-regulation of type I interferons (IFNs), we hypothesized that the strong IL-12-inducing capacity of Lactobacillus acidophilus NCFM in murine bone-marrow-derived dendritic cells (DCs) is caused by an up-regulation of IFN-β, which subsequently induces IL-12 and the double-stranded RNA binding Toll-like receptor-3 (TLR-3). The expression of the genes encoding IFN-β, TLR-3, IL-12 and IL-10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn-β, Il-12 and Il-10 compared with the synthetic double-stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr-3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up-regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN-β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN-β expression was markedly reduced in TLR-2(-/-) DCs, dependent on endocytosis, and the major cause of the induction of Il-12 and Tlr-3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR-2 manner dependent on IFN-β.

  18. Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism

    PubMed Central

    Weiss, Gudrun; Rasmussen, Simon; Zeuthen, Louise Hjerrild; Nielsen, Birgit Nøhr; Jarmer, Hanne; Jespersen, Lene; Frøkiær, Hanne

    2010-01-01

    Lactobacilli are probiotics that, among other health-promoting effects, have been ascribed immunostimulating and virus-preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin-12 (IL-12) -inducing properties. As IL-12 production depends on the up-regulation of type I interferons (IFNs), we hypothesized that the strong IL-12-inducing capacity of Lactobacillus acidophilus NCFM in murine bone-marrow-derived dendritic cells (DCs) is caused by an up-regulation of IFN-β, which subsequently induces IL-12 and the double-stranded RNA binding Toll-like receptor-3 (TLR-3). The expression of the genes encoding IFN-β, TLR-3, IL-12 and IL-10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn-β, Il-12 and Il-10 compared with the synthetic double-stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr-3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up-regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN-β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN-β expression was markedly reduced in TLR-2−/− DCs, dependent on endocytosis, and the major cause of the induction of Il-12 and Tlr-3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR-2 manner dependent on IFN-β. PMID:20545783

  19. Gene expression profiles of hair and wool sheep reveal importance of Th2 immune mechanisms for increased resistance to.

    PubMed

    MacKinnon, K M; Bowdridge, S A; Kanevsky-Mullarky, I; Zajac, A M; Notter, D R

    2015-05-01

    Management of gastrointestinal parasites is a critical issue for sheep producers worldwide. Increases in the prevalence of drug-resistant worms have complicated parasite control and increased economic losses. Therefore, other methods of parasite control need to be assessed, including the use of genetically resistant animals in breeding programs. Hair sheep breeds such as the St. Croix have greater parasite resistance than conventional wool breeds. However, the immune mechanisms that control parasite resistance in hair or wool breeds have not yet been fully determined, and information on cytokine expression profiles for both wool sheep selected for increased resistance and hair sheep is limited. Our objective was to investigate gene expression differences in 24 parasite-resistant hair and 24 susceptible wool sheep to identify immune effectors associated with resistance to . One-half of the lambs were infected and sacrificed at 3 or 27 d after infection. Remaining lambs were not infected. Breed differences in expression of genes associated with Th1 and Th2 immune responses in lymph nodes and abomasal tissue were determined. Th2-associated genes included IL-4, IL-13, IL-5, IgE, the α chain of the IL-4 receptor, and the α chain of the high-affinity IgE receptor (FcεRI). Th1-associated genes included interferon gamma (IFN-γ), the p35 subunit of IL-12 (IL-12 p35), and the β1 and β2 chains of the IL-12 receptor (IL-12 Rβ1 and IL-12 Rβ2, respectively). In both hair and wool sheep, infection with resulted in greater expression of IgE, IL-13, IL-5, and IL-12 p35 and somewhat reduced expression of IFNγ in lymph nodes. In abomasal tissue, parasite infection resulted in greater IgE, IL-13, FcεRI, and IL-12 p35 expression in infected lambs compared with control lambs. Between breeds, hair sheep had a stronger Th2 response after infection than wool sheep, with increased expression of IgE and IL-13 and decreased expression of IFNγ in lymph nodes and increased expression

  20. The importance of local mucosal HIV-specific CD8(+) cytotoxic T lymphocytes for resistance to mucosal viral transmission in mice and enhancement of resistance by local administration of IL-12.

    PubMed Central

    Belyakov, I M; Ahlers, J D; Brandwein, B Y; Earl, P; Kelsall, B L; Moss, B; Strober, W; Berzofsky, J A

    1998-01-01

    Although crucial to mucosal vaccine development, the mechanisms of defense against mucosal viral infection are still poorly understood. Protection, cytotoxic T lymphocytes (CTL), and neutralizing antibodies have all been observed, but cause and effect have been difficult to determine. The ability of CTL in the mucosa to mediate protection against mucosal viral transmission has never been proven. Here, we use an HIV peptide immunogen and an HIV-1 gp160-expressing recombinant vaccinia viral intrarectal murine challenge system, in which neutralizing antibodies do not play a role, to demonstrate for the first time that long-lasting immune resistance to mucosal viral transmission can be accomplished by CD8(+) CTL that must be present in the mucosal site of exposure. The resistance is ablated by depleting CD8(+) cells in vivo and requires CTL in the mucosa, whereas systemic (splenic) CTL are shown to be unable to protect against mucosal challenge. Furthermore, the resistance as well as the CTL response can be increased by local mucosal delivery of IL-12 with the vaccine. These results imply that induction of local mucosal CTL may be critical for success of a vaccine against viruses transmitted through a mucosal route, such as HIV. PMID:9854042

  1. HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses in fractionated γ-irradiated mice by modulating the IL-12p70-STAT4 signaling pathway.

    PubMed

    Park, Hae-Ran; Jo, Sung-Kee; Choi, Nam-Hee; Jung, Uhee

    2012-05-01

    Whole body irradiated mice appear to experience a down-regulation of the helper T (Th)1-like immune response, and maintain a persistent immunological imbalance. In the current study, we evaluated the effect of HemoHIM (an herbal product made from Angelica Radix, Cnidium officinale , and Paeonia japonica cultivated in Korea) to ameliorate the immunological imbalance induce in fractionated γ-irradiated mice. The mice were exposed to γ rays twice a week (0.5 Gy fractions) for a total dose of 5 Gy, and HemoHIM was administrated orally from 1 week before the first irradiation to 1 week before the final analysis. All experiments were performed 4 and 6 months after their first exposure. HemoHIM ameliorated the Th1- and Th2-related immune responses normally occur in irradiated mice with or without dinitrophenylated keyhole limpet hemocyanin immunization. HemoHIM also restored the natural killer cell activities without changing the percentage of natural killer cells in irradiated mice. Furthermore, the administration of HemoHIM prevented the reduction in levels of interleukin-12p70 in irradiated mice. Finally, we found that HemoHIM enhanced the phosphorylation of signal transducer and activator of transcription (STAT) 4 that was reduced in irradiated mice. Our findings suggest that HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses by modulating the IL-12p70/pSTAT4 signaling pathway.

  2. Submerged cultivation of Ganoderma lucidum and the effects of its polysaccharides on the production of human cytokines TNF-α, IL-12, IFN-γ, IL-2, IL-4, IL-10 and IL-17.

    PubMed

    Habijanic, Jožica; Berovic, Marin; Boh, Bojana; Plankl, Mojca; Wraber, Branka

    2015-01-25

    An original strain of Ganoderma lucidum (W.Curt.:Fr.) Lloyd, MZKI G97 isolated from Slovenian habitats was grown by a submerged liquid substrate cultivation in a laboratory stirred tank reactor. Five fractions of extracellular and cell-wall polysaccharides were obtained by extraction, ethanol precipitation, and purification by ion-exchange, gel and affinity chromatography. The capacity of isolated polysaccharide fractions to induce innate inflammatory cytokines, and to modulate cytokine responses of activated lymphocytes was investigated. Human peripheral blood mononuclear cells (PBMC) were activated in vitro with polysaccharide fractions, in order to induce innate inflammatory cytokines: tumor necrosis factor alpha (TNF-α), interleukin (IL) 12 and interferon gamma (IFN-γ). For the immunomodulation capacity, polysaccharide fractions were cultured with ionomycine and phorbol myristate acetate (IONO+PMA) activated PBMC, and the concentrations of induced IL-2, IL-4, IFN-γ, IL-10 and IL-17 were measured. The results showed that polysaccharides from G. lucidum induced moderate to high amounts of innate inflammatory cytokines. Fungal cell-wall polysaccharides were stronger innate inflammatory cytokines inducers, while extracellular polysaccharides demonstrated a higher capacity to modulate cytokine responses of IONO+PMA induced production of IL-17. The results indicate that G. lucidum polysaccharides enhance Th1 response with high levels of IFN-γ and IL-2, and display low to no impact on IL-4 production. A similar pattern was observed at regulatory cytokine IL-10. All of the polysaccharide fractions tested induced IL-17 production at different concentration levels.

  3. [Two siblings with an IL-12 and IFN-γ production disorder diagnosed with pulmonary mycobacteriosis caused by M. kansasii. Mendelian susceptibility to mycobacterial infection: an overview of literature].

    PubMed

    Nalepa, Piotr; Strach, Magdalena; Rybak-Bąk, Marta; Siedlar, Maciej

    2011-01-01

    Two previously healthy siblings were diagnosed with pulmonary mycobacteriosis caused by M. kansasii. During examination both patients were diagnosed with an interleukin 12 (IL-12) and interferon γ (IFN-γ) production disorder of the stimulated lymphocytes. The given genetic defect conditions the occurrence of the Mendelian susceptibility to mycobacterial infection (MSMD). The patients fulfilled clinical, radiological, and bacteriological criteria for diagnosis of mycobacteriosis laid out by American Thoracic Society in 2007. After 13 months of standard treatment the ailments receded, and radiological remission, as well as a 12-month-lasting sputum negativity was achieved. The prognosis for the patients remains uncertain. The genetic conditioning to mycobacterial infections may cause disease recurrences or other mycobacterial illnesses. The patients will need to be checked systematically by pulmonologist. It is not known whether the offspring of the patients are exposed to general Baccillus Calmette-Guérin infection due to the compulsory vaccinations against tuberculosis, and whether the risk of complications is higher than the potential risk of coming down with hematogenous TB in childhood.

  4. Cloning and sequence analysis of Peromyscus yucatanicus (Rodentia) Th1 (IL-12p35, IFN-γ and TNF) and Th2 (IL-4, IL-10 and TGF-β) cytokines.

    PubMed

    Loria-Cervera, Elsy Nalleli; Sosa-Bibiano, Erika Ivett; Villanueva-Lizama, Liliana Estefania; Van Wynsberghe, Nicole Raymonde; Schountz, Tony; Andrade-Narvaez, Fernando Jose

    2014-01-01

    The Yucatan deer mouse, Peromyscus yucatanicus (order Rodentia), is the principal reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico. Experimental infection results in clinical and histopathological features similar to those observed in humans with cutaneous leishmaniasis (CL) as well as peritoneal macrophage production of nitric oxide. These results support the possible use of P. yucatanicus as a novel experimental model to study CL caused by L. (L.) mexicana. However, immunological studies in these rodents have been limited by the lack of specific reagents. To address this issue, we cloned and analyzed cytokine sequences of P. yucatanicus as part of an effort to develop this species as a CL model. We cloned P. yucatanicus interleukin 4 (IL-4), IL-10, IL-12p35, gamma interferon, transforming growth factor beta and tumor necrosis factor partial cDNAs. Most of the P. yucatanicus sequences were highly conserved with orthologs of other mammalian species and the identity of all sequences were confirmed by the presence of conserved amino acids with possible biological functions in each putative polypeptide. The availability of these sequences is a first step which will allow us to carry out studies characterizing the immune response during pathogenic and nonpathogenic L. (L.) mexicana infections in P. yucatanicus. Copyright © 2013. Published by Elsevier Ltd.

  5. Resequencing candidate genes implicates rare variants in asthma susceptibility.

    PubMed

    Torgerson, Dara G; Capurso, Daniel; Mathias, Rasika A; Graves, Penelope E; Hernandez, Ryan D; Beaty, Terri H; Bleecker, Eugene R; Raby, Benjamin A; Meyers, Deborah A; Barnes, Kathleen C; Weiss, Scott T; Martinez, Fernando D; Nicolae, Dan L; Ober, Carole

    2012-02-10

    Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Resequencing Candidate Genes Implicates Rare Variants in Asthma Susceptibility

    PubMed Central

    Torgerson, Dara G.; Capurso, Daniel; Mathias, Rasika A.; Graves, Penelope E.; Hernandez, Ryan D.; Beaty, Terri H.; Bleecker, Eugene R.; Raby, Benjamin A.; Meyers, Deborah A.; Barnes, Kathleen C.; Weiss, Scott T.; Martinez, Fernando D.; Nicolae, Dan L.; Ober, Carole

    2012-01-01

    Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10−4) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease. PMID:22325360

  7. Enhanced Delivery of Plasmid Encoding Interleukin-12 Gene by Diethylene Triamine Penta-Acetic Acid (DTPA)-Conjugated PEI Nanoparticles.

    PubMed

    Dehshahri, Ali; Sadeghpour, Hossein; Keykhaee, Maryam; Khalvati, Bahman; Sheikhsaran, Fatemeh

    2016-05-01

    Recombinant therapeutic proteins have been considered as an efficient category of medications used for the treatment of various diseases. Despite their effectiveness, there are some reports on the systemic adverse effects of recombinant therapeutic proteins limiting their wide clinical applications. Among different cytokines used for cancer immunotherapy, interleukin-12 (IL-12) has shown great ability as a powerful antitumor and antiangiogenic agent. However, significant toxic reactions following the systemic administration of IL-12 have led researchers to seek for alternative approaches such as the delivery and local expression of the IL-12 gene inside the tumor tissues. In order to transfer the plasmid encoding IL-12 gene, the most extensively investigated polycationic polymer, polyethylenimine (PEI), was modified by diethylene triamine penta-acetic acid (DTPA) to modulate the hydrophobic-hydrophilic balance of the polymer as well as its toxicity. DTPA-conjugated PEI derivatives were able to form complexes in the size range around 100-180 nm with great condensation ability and protection of the plasmid against enzymatic degradation. The highest gene transfer ability was achieved by the DTPA-conjugated PEI at the conjugation degree of 0.1 % where the level of IL-12 production increased up to twofold compared with that of the unmodified PEI. Results of the present study demonstrated that modulation of the surface positive charge of PEI along with the improvement of the polymer hydrophobic balance could be considered as a successful strategy to develop safe and powerful nanocarriers.

  8. Clinical Impact of Immune Microenvironment in Stage I Lung Adenocarcinoma: Tumor Interleukin-12 Receptor β2 (IL-12Rβ2), IL-7R, and Stromal FoxP3/CD3 Ratio Are Independent Predictors of Recurrence

    PubMed Central

    Suzuki, Kei; Kadota, Kyuichi; Sima, Camelia S.; Nitadori, Jun-ichi; Rusch, Valerie W.; Travis, William D.; Sadelain, Michel; Adusumilli, Prasad S.

    2013-01-01

    Purpose Mounting evidence suggests that tumor-infiltrating immune cells have prognostic value for patients with solid organ malignancies. Our aim was to investigate the prognostic significance of the immune microenvironment in patients with stage I lung adenocarcinoma (ADC). Patients and Methods Using tissue microarray and immunohistochemistry, we investigated eight types of tumor-infiltrating immune cells in the tumor nest and tumor-associated stroma as well as tumor expression of five cytokines in a uniform cohort of 956 patients with stage I lung ADC (478 each in training and validation cohorts). Results Although a high density of stromal forkhead box P3 (FoxP3) –positive cells was associated with shorter recurrence-free probability (RFP; P = .043), the relative proportion of stromal FoxP3 to CD3 was a stronger predictor of recurrence (5-year RFP, 85% for high v 77% for low ratio; P = .004). High expression of tumor interleukin-12 receptor β2 (IL-12Rβ2) was associated with better outcome (5-year RFP, 90% for high v 80% for low expression; P = .026), whereas high expression of tumor IL-7R was associated with worse outcome (5-year RFP, 76% for high v 86% for low expression; P = .001). In multivariate analysis, these immune markers were independently associated with recurrence. Although IL-7R remained significant for poor overall survival, all the markers remained prognostic for recurrence in patients with stages IA and IB disease as well as for patients with tumors ≤ 2 cm. Conclusion Our investigation confirms the biologic and prognostic significance of the tumor immune microenvironment for patients with stage I lung ADC and provides support for its use to stratify clinical outcome and immunotherapeutic interventions. PMID:23269987

  9. Withania somnifera chemotype NMITLI 101R significantly increases the efficacy of antileishmanial drugs by generating strong IFN-γ and IL-12 mediated immune responses in Leishmania donovani infected hamsters.

    PubMed

    Tripathi, Chandra Dev Pati; Kushawaha, Pramod Kumar; Sangwan, Rajender Singh; Mandal, Chitra; Misra-Bhattacharya, Shailja; Dube, Anuradha

    2017-01-15

    Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems. Pharmacological studies have established that root extracts of W. somnifera contain several bioactive constituents called withanolides. The plant has long been used for its several beneficial properties and recently as an immunomodulator. A combination therapy including a potential and safe immunostimulant with lower doses of effective drug, which can reduce the parasitic burden and simultaneously can produce an enhancement of adaptive immunity, has proven to be significantly a more effective approach than immunotherapy or drug therapy alone. Evaluation of the immunostimulatory effect of W. somnifera chemotype NMITLI 101R when used in combination with ED50 doses of antileishmanial drugs in Leishmania donovani infected hamsters. Infected animals were administered with chemotype 101R(30mg/kg × 15 days) either alone or in combination with ED50 doses of miltefosine (10mg/kg × 5 days), paromomycin (30mg/kg × 5 days) or amphotericin B (0.5mg/kg × 5 days). The treated animals were euthanized on days 30 and 60 post-treatment (p.t.) and checked for parasite clearance, delayed type hypersensitivity (DTH) response, cytokine and inducible nitric oxide synthase levels by real-time PCR, nitric oxide (NO) production, reactive oxygen species (ROS) generation, lymphoproliferative and antibody responses. The group of animals that received 101R and ED50 dose of miltefosine showed optimum inhibition of parasite multiplication (∼98%) by day 60 p.t. followed by the group that received 101R plus paromomycin (∼94%) and 101R plus amphotericin B (∼93%). The efficacy was well supported by the increased inducible NO synthase mRNA transcript, strong IFN-γand IL-12 mediated Th1 immune responses and significantly suppressed levels of Th2 cytokines (IL-4, IL-10 and TGF-β). Additionally, same therapy

  10. Expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine.

    PubMed

    Akhzari, Soheyla; Rezvan, Hossein; Zolhavarieh, Seyed Masoud

    2017-07-01

    It is now supposed that cytokines released during the burn injuries have a great impact on the immunological and pathological responses after the burn. The main objective of this study was to investigate the expression of some pro-inflammatory genes in the wound, spleen and blood neutrophils during the healing process of burn wounds in a murine model. The expression of ten pro-inflammatory genes were examined in wounds, spleens and blood neutrophils of mice with burn injuries treated with either silver sulfodiazine or phosphate-buffered saline (PBS) using RT-PCR at the end of the first and second weeks after injuries. None of the pro-inflammatory genes were expressed in the skin, spleen and blood neutrophils of healthy mice. In the group control, IL-12P35, IL-12P40, CCR5, IL-1β and IFN-γ were expressed in the spleen and blood neutrophils in the first week. Instead, CCL5, CCR5, IL-1β and IFN-γ were expressed in the wound, but in the second week, the expression of the genes became similar. In the test group, in the first week, TNF-α, IL-12P35, IL-12P40 and IL-1β were expressed in the lesions, CCL4, IL-1α, IL-12P35, IL-12P40, CCR5 and IFN-γ were expressed in the spleen and no pro-inflammatory gene expression was detected in blood neutrophils. IL-1β and IFN-γ are expressed in wound, spleen and neutrophils of untreated mice, but not in silver sulfodiazine treated mice. Hence, treatment with silver sulfodiazine suppressed the expression of pro-inflammatory genes in some stages of healing.

  11. Cancer gene therapy utilized ultrasound (US)-sensitive liposome as non-viral vector

    NASA Astrophysics Data System (ADS)

    Suzuki, Ryo; Oda, Yusuke; Namai, Eisuke; Nishiie, Norihito; Hirata, Keiichi; Taira, Yuichiro; Utoguchi, Naoki; Negichi, Yoichi; Maruyama, Kazuo

    2010-03-01

    Sonoporation is an attractive technique to develop non-invasive and non-viral gene delivery system. However, simple sonoporation using only ultrasound (US) is not enough to establish effective cancer gene therapy because of low efficiency of gene delivery. Therefore, we improved this problem by the combination of US and novel US-sensitive liposome (Bubble liposome) which was a liposome containing US imaging gas (perfluoropropane). This was an effective gene delivery system with collapse (cavitation) that was induced by US exposure to Bubble liposome. In this study, we assessed the ability of this system in cancer gene therapy using IL-12 cording plasmid DNA. The combination of Bubble liposomes and ultrasound was dramatically suppressed tumor growth. Therefore, we concluded that the combination of Bubble liposomes and ultrasound would be a good non-viral vector system in IL-12 cancer gene therapy.

  12. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery.

    PubMed

    Best, Simon R; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R; Wu, T-C; Pai, Sara I

    2009-09-04

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination--conventional intramuscular injection, electroporation-mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery--in the ability to generate antigen-specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin's role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials.

  13. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

    PubMed Central

    Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

    2009-01-01

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

  14. Cytokine gene-mediated immunotherapy: current status and future perspectives.

    PubMed

    Jinushi, Masahisa; Tahara, Hideaki

    2009-08-01

    Recent understanding of the molecular events crucial in overcoming immunosuppressive tumor microenvironments and generating effective antitumor immunity provides us with the wreath opportunity to manipulate genes that have a key role in antitumor immune responses. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) are two indispensable cytokines for activating dendritic cells and boosting the strong immune responses against cancer. In this review, we describe the antitumor mechanisms and clinical application of gene-modified tumor cells and dendritic cells to secrete GM-CSF or IL-12, respectively, in various preclinical and clinical settings. The principles operative in these vaccination strategies may prove applicable to other immunotherapy strategies, especially in combination with other therapeutic modalities, such as chemotherapy and targeted therapy.

  15. Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment.

    PubMed

    Tillack, Cornelia; Ehmann, Laura Maximiliane; Friedrich, Matthias; Laubender, Rüdiger P; Papay, Pavol; Vogelsang, Harald; Stallhofer, Johannes; Beigel, Florian; Bedynek, Andrea; Wetzke, Martin; Maier, Harald; Koburger, Maria; Wagner, Johanna; Glas, Jürgen; Diegelmann, Julia; Koglin, Sarah; Dombrowski, Yvonne; Schauber, Jürgen; Wollenberg, Andreas; Brand, Stephan

    2014-04-01

    We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor α (TNF-α) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD). Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab. Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-γ-secreting Th1 cells and IFN-α-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab. New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL-23 antibody therapy is a highly effective therapy for these lesions.

  16. Combination electro-gene therapy using herpes virus thymidine kinase and interleukin-12 expression plasmids is highly efficient against murine carcinomas in vivo.

    PubMed

    Goto, Tomoaki; Nishi, Toru; Kobayashi, Osamu; Tamura, Takahiko; Dev, Sukhendu B; Takeshima, Hideo; Kochi, Masato; Kuratsu, Jun-ichi; Sakata, Tsuneaki; Ushio, Yukitaka

    2004-11-01

    We report the use of plasmid DNA-mediated combination gene therapy for tumor-bearing mice using in vivo electroporation, also called electro-gene therapy (EGT), that resulted in uncomplicated and complete cures in more than 90% of the mice. Subcutaneously inoculated CT26 tumors in syngeneic BALB/c mice were subjected to repeated EGT treatments consisting of intratumoral co-injection of naked plasmids encoding the cytokine interleukin-12 (IL-12) (p35 and p40 subunits) and the suicide gene herpes simplex virus thymidine kinase (HSV-tk), followed by in vivo electroporation. The early anti-tumor effect was always stronger, and the rate of cure, as seen in the long-term follow-up, was always greater in the groups treated with combination EGT than in those treated with IL-12 or HSV-tk EGT alone. Systemic levels of IL-12 and IFN-gamma increased in both combination and IL-12-alone EGT-treated groups. Moreover, combination EGT for established subcutaneous tumors strongly reduced hematogenous lung metastases and increased survival time when live CT26 tumor cells were injected through the tail vein. Limited experiments on C57/B16 mice with murine melanoma also showed very similar trends. These results suggest that this simple and safe method of plasmid-mediated combination EGT may provide a potentially effective gene therapy for cancer.

  17. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  18. Prospects for herpes-simplex-virus thymidine-kinase and cytokine gene transduction as immunomodulatory gene therapy for prostate cancer.

    PubMed

    Hassan, W; Sanford, M A; Woo, S L; Chen, S H; Hall, S J

    2000-04-01

    In completed and ongoing clinical trials, adenovirus-mediated (Ad.) expression of herpes-simplex-virus thymidine-kinase (HSV-tk) gene transduction followed by ganciclovir (GCV) therapy has produced limited toxicity and evidence of antitumor activity following injection of the prostate. Furthermore, this system has been shown to direct systemic antitumor activity in several experimental cancer models, including that of prostate cancer, which may serve as the basis for in-situ immunomodulatory gene therapy. In a mouse model of prostate cancer, natural killer (NK) cells have been identified as the mediator of antimetastatic activity following Ad.HSV-tk + GCV, resulting in the combination of Ad.HSV-tk and adenovirus-mediated expression of interleukin 12 (Ad.IL-12) to exploit this cytokine's ability to enhance NK proliferation and cytotoxicity. Combination therapy demonstrated superior local and systemic growth suppression over that obtained with either therapy alone. Importantly, when the metastatic tumor burden was increased to an extent that negated the growth-suppressive activity directed by Ad.HSV-tk + GCV or Ad.IL-12 alone, combination therapy continued to demonstrate significant growth suppression. Examination of tumor-infiltrating lymphocytes documented enhanced NK lytic activity following combination therapy. Therefore, it appears that the combination of Ad.HSV-tk and Ad.IL-12 should be validated in a clinical trial for the treatment of prostate cancer.

  19. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

    PubMed Central

    2010-01-01

    Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs). Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti

  20. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  1. Augmentation of a Novel Enzyme/Pro-Drug Gene Therapy "Distant Bystander Effect" to Target Prostate Cancer Metastasis

    DTIC Science & Technology

    2005-09-01

    enzyme/prodrug therapy(GDEPT)_ mIL-12; mIL-18; cytosine deaminase and uracil phopho-ribosyl transferase (CDUPRT) 16. SECURITY CLASSIFICATION OF: 17...therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase (CD/UPRT)) is delivered to a cancer cell so that harmless bacterial...reduction both at the treatment site and at remote locations. In this therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase

  2. Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Thakur, Aneesh; Riber, Ulla; Davis, William C; Jungersen, Gregers

    2013-10-01

    T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag-specific IFN-γ production by ELISA and flow cytometry. There was a significant increase in production of IFN-γ by T cell subsets or NKp46+ cells cultured in the presence of Ag and aCD28/aCD49d. The increase was accompanied by an increase in the integrated median fluorescence intensity (iMFI) of activated T cells. Addition of rIL-12 induced a significant additive effect leading to a maximum increase in responder frequency of Ag-specific T cell subsets or NKp46+ cells with a heavy bias toward IFN-γ production by CD4 T cells. We provide the first description of using aCD28/aCD49d costimulation to potentiate an Ag-specific increase in the production of IFN-γ in bovine immunology. The study also shows the degree of signaling in T cells is regulated by the costimulatory environment.

  3. The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation

    PubMed Central

    2010-01-01

    Background High levels of ascorbic acid (AsA) in tomato fruits provide health benefits for humans and also play an important role in several aspects of plant life. Although AsA metabolism has been characterized in detail, the genetic mechanisms controlling AsA accumulation in tomatoes are poorly understood. The transcriptional control of AsA levels in fruits can be investigated by combining the advanced genetic and genomic resources currently available for tomato. A comparative transcriptomic analysis of fruit tissues was carried out on an introgression line containing a QTL promoting AsA accumulation in the fruit, using a parental cultivar with lower AsA levels as a reference. Results Introgression line IL 12-4 (S. pennellii in a S. lycopersicum background) was selected for transcriptomic analysis because it maintained differences in AsA levels compared to the parental genotypes M82 and S. pennellii over three consecutive trials. Comparative microarray analysis of IL 12-4 and M82 fruits over a 2-year period allowed 253 differentially-expressed genes to be identified, suggesting that AsA accumulation in IL 12-4 may be caused by a combination of increased metabolic flux and reduced utilization of AsA. In particular, the upregulation of a pectinesterase and two polygalacturonases suggests that AsA accumulation in IL12-4 fruit is mainly achieved by increasing flux through the L-galactonic acid pathway, which is driven by pectin degradation and may be triggered by ethylene. Conclusions Based on functional annotation, gene ontology classification and hierarchical clustering, a subset of the 253 differentially-expressed transcripts was used to develop a model to explain the higher AsA content in IL 12-4 fruits in terms of metabolic flux, precursor availability, demand for antioxidants, abundance of reactive oxygen species and ethylene signaling. PMID:20691085

  4. Classification of dendritic cell phenotypes from gene expression data

    PubMed Central

    2011-01-01

    Background The selection of relevant genes for sample classification is a common task in many gene expression studies. Although a number of tools have been developed to identify optimal gene expression signatures, they often generate gene lists that are too long to be exploited clinically. Consequently, researchers in the field try to identify the smallest set of genes that provide good sample classification. We investigated the genome-wide expression of the inflammatory phenotype in dendritic cells. Dendritic cells are a complex group of cells that play a critical role in vertebrate immunity. Therefore, the prediction of the inflammatory phenotype in these cells may help with the selection of immune-modulating compounds. Results A data mining protocol was applied to microarray data for murine cell lines treated with various inflammatory stimuli. The learning and validation data sets consisted of 155 and 49 samples, respectively. The data mining protocol reduced the number of probe sets from 5,802 to 10, then from 10 to 6 and finally from 6 to 3. The performances of a set of supervised classification models were compared. The best accuracy, when using the six following genes --Il12b, Cd40, Socs3, Irgm1, Plin2 and Lgals3bp-- was obtained by Tree Augmented Naïve Bayes and Nearest Neighbour (91.8%). Using the smallest set of three genes --Il12b, Cd40 and Socs3-- the performance remained satisfactory and the best accuracy was with Support Vector Machine (95.9%). These data mining models, using data for the genes Il12b, Cd40 and Socs3, were validated with a human data set consisting of 27 samples. Support Vector Machines (71.4%) and Nearest Neighbour (92.6%) gave the worst performances, but the remaining models correctly classified all the 27 samples. Conclusions The genes selected by the data mining protocol proposed were shown to be informative for discriminating between inflammatory and steady-state phenotypes in dendritic cells. The robustness of the data mining

  5. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    PubMed Central

    Weiss, Gudrun; Rasmussen, Simon; Nielsen Fink, Lisbeth; Jarmer, Hanne; Nøhr Nielsen, Birgit; Frøkiær, Hanne

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-β, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate immune response. Moreover, B. bifidum Z9 inhibited the expression of the Th1-promoting genes induced by L. acidophilus NCFM and had an additive effect on genes of the innate immune response and Th2 skewing genes. The gene encoding Jun dimerization protein 2 (JDP2), a transcription factor regulating the activation of JNK, was one of the few genes only induced by B. bifidum Z9. Neutralization of IFN-β abrogated L. acidophilus NCFM-induced expression of Th1-skewing genes, and blocking of the JNK pathway completely inhibited the expression of IFN-β. Our results indicate that B. bifidum Z9 actively inhibits the expression of genes related to the adaptive immune system in murine dendritic cells and that JPD2 via blocking of IFN-β plays a central role in this regulatory mechanism. PMID:20548777

  6. Systems biology of IL-6, IL-12 family cytokines.

    PubMed

    Dittrich, Anna; Hessenkemper, Wiebke; Schaper, Fred

    2015-10-01

    Interleukin-6-type cytokines play important roles in the communication between cells of multicellular organisms. They are involved in the regulation of complex cellular processes such as proliferation and differentiation and act as key player during inflammation and immune response. A major challenge is to understand how these complex non-linear processes are connected and regulated. Systems biology approaches are used to tackle this challenge in an iterative process of quantitative experimental and mathematical analyses. Here we review quantitative experimental studies and systems biology approaches dealing with the function of Interleukin-6-type cytokines in physiological and pathophysiological conditions. These approaches cover the analyses of signal transduction on a cellular level up to pharmacokinetic and pharmacodynamic studies on a whole organism level.

  7. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    SciTech Connect

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  8. Regulation of signal transducer and activator of transcription and suppressor of cytokine-signaling gene expression in the brain of mice with astrocyte-targeted production of interleukin-12 or experimental autoimmune encephalomyelitis.

    PubMed

    Maier, Joachim; Kincaid, Carrie; Pagenstecher, Axel; Campbell, Iain L

    2002-01-01

    Interleukin (IL)-12 and interferon (IFN)-gamma are implicated in the pathogenesis of immune disorders of the central nervous system (CNS). To define the basis for the actions of these cytokines in the CNS, we examined the temporal and spatial regulation of key signal transducers and activators of transcription (STATs) and suppressors of cytokine signaling (SOCS) in the brain of transgenic mice with astrocyte production of IL-12 or in mice with experimental autoimmune encephalomyelitis (EAE). In healthy mice, with the exception of STAT4 and STAT6, the expression of a number of STAT and SOCS genes was detectable. However, in symptomatic transgenic mice and in EAE significant up-regulation of STAT1, STAT2, STAT3, STAT4, IRF9, and SOCS1 and SOCS3 RNA transcripts was observed. Although the increased expression of STAT1 RNA was widely distributed and included neurons, astrocytes, and microglia, STAT4 and STAT3 and SOCS1 and SOCS3 RNA was primarily restricted to the infiltrating mononuclear cell population. The level and location of the STAT1, STAT3, and STAT4 proteins overlapped with their corresponding RNA and additionally showed nuclear localization indicative of activation of these molecules. Thus, in both the glial fibrillary acidic protein-IL-12 mice and in EAE the CNS expression of key STAT and SOCS genes that regulate IL-12 (STAT4) and IFN-gamma (STAT1, SOCS1, and SOCS3) receptor signaling is highly regulated and compartmentalized. We conclude the interaction between these positive and negative signaling circuits and their distinct cellular locations likely play a defining role in coordinating the actions of IL-12 and IFN-gamma during the pathogenesis of type 1 immune responses in the CNS.

  9. Regulation of Signal Transducer and Activator of Transcription and Suppressor of Cytokine-Signaling Gene Expression in the Brain of Mice with Astrocyte-Targeted Production of Interleukin-12 or Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Maier, Joachim; Kincaid, Carrie; Pagenstecher, Axel; Campbell, Iain L.

    2002-01-01

    Interleukin (IL)-12 and interferon (IFN)-γ are implicated in the pathogenesis of immune disorders of the central nervous system (CNS). To define the basis for the actions of these cytokines in the CNS, we examined the temporal and spatial regulation of key signal transducers and activators of transcription (STATs) and suppressors of cytokine signaling (SOCS) in the brain of transgenic mice with astrocyte production of IL-12 or in mice with experimental autoimmune encephalomyelitis (EAE). In healthy mice, with the exception of STAT4 and STAT6, the expression of a number of STAT and SOCS genes was detectable. However, in symptomatic transgenic mice and in EAE significant up-regulation of STAT1, STAT2, STAT3, STAT4, IRF9, and SOCS1 and SOCS3 RNA transcripts was observed. Although the increased expression of STAT1 RNA was widely distributed and included neurons, astrocytes, and microglia, STAT4 and STAT3 and SOCS1 and SOCS3 RNA was primarily restricted to the infiltrating mononuclear cell population. The level and location of the STAT1, STAT3, and STAT4 proteins overlapped with their corresponding RNA and additionally showed nuclear localization indicative of activation of these molecules. Thus, in both the glial fibrillary acidic protein-IL-12 mice and in EAE the CNS expression of key STAT and SOCS genes that regulate IL-12 (STAT4) and IFN-γ (STAT1, SOCS1, and SOCS3) receptor signaling is highly regulated and compartmentalized. We conclude the interaction between these positive and negative signaling circuits and their distinct cellular locations likely play a defining role in coordinating the actions of IL-12 and IFN-γ during the pathogenesis of type 1 immune responses in the CNS. PMID:11786421

  10. Adenovirus-mediated tumor-specific combined gene therapy using Herpes simplex virus thymidine/ganciclovir syste