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Sample records for interleukin-12 p40 homodimer

  1. IL-12p40 Homodimer Ameliorates Experimental Autoimmune Arthritis

    PubMed Central

    Lee, Seon-Yeong; Jung, Young Ok; Kim, Doo-Jin; Kang, Chang-Min; Moon, Young-Mee; Heo, Yu-Jung; Oh, Hye-Jwa; Park, Seong-Jeong; Yang, Se-Hwan; Kwok, Seung Ki; Ju, Ji-Hyeon; Park, Sung-Hwan; Sung, Young Chul

    2015-01-01

    IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist–knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4+CD25+Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor–related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling. PMID:26324771

  2. IL-12p40 Homodimer Ameliorates Experimental Autoimmune Arthritis.

    PubMed

    Lee, Seon-Yeong; Jung, Young Ok; Kim, Doo-Jin; Kang, Chang-Min; Moon, Young-Mee; Heo, Yu-Jung; Oh, Hye-Jwa; Park, Seong-Jeong; Yang, Se-Hwan; Kwok, Seung Ki; Ju, Ji-Hyeon; Park, Sung-Hwan; Sung, Young Chul; Kim, Ho-Youn; Cho, Mi-La

    2015-10-01

    IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist-knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4(+)CD25(+)Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor-related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling. PMID:26324771

  3. Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-γ stimulated macrophages

    PubMed Central

    Yamauchi, Keiko; Shibata, Yoko; Kimura, Tomomi; Abe, Shuichi; Inoue, Sumito; Osaka, Daisuke; Sato, Michiko; Igarashi, Akira; Kubota, Isao

    2009-01-01

    Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-γ stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-γ. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-γ in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-γ-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages. PMID:19893639

  4. Inflammation and Elevation of Interleukin-12p40 in Patients with Schizophrenia.

    PubMed

    Bedrossian, Nora; Haidar, Mariam; Fares, Jawad; Kobeissy, Firas H; Fares, Youssef

    2016-01-01

    Schizophrenia is a serious mental illness with chronic symptoms and significant impairment in psychosocial functioning, which suggests that it likely has neurodegenerative characteristics. Inflammatory markers such as pro-inflammatory cytokines are well-known etiological contributors for psychiatric disorders, including schizophrenia. Although, the role of inflammation in schizophrenia is becoming evident, the number of studies in this area is relatively scarce, especially in Lebanon, and increased procedural thoroughness is needed. Cytokines play a key role in the activation of the immune system and strongly influence neurotransmission. Previous investigation of plasma levels showed dysregulation of interleukin (IL)-12. However, genotypical variations of this interleukin have not been investigated for patients with schizophrenia yet. Thus, in this paper, we aimed to compute and assess IL-12p40 levels in the sera of individuals with schizophrenia from different provinces in Lebanon and compare it to controls. Healthy subjects comprised 60 individuals with a male/female (M/F) ratio of 31/29, whereas patients with schizophrenia consisted of 63 subjects with an M/F ratio of 30/33. The mean age for healthy controls was 30 years, whereas that for patients with schizophrenia was 35 years. A standardized enzyme-linked immunosorbent assay (ELISA) technique was used to measure the concentration of IL-12p40 in all collected sera (n = 123). The mean IL-12p40 levels in patients with schizophrenia were significantly higher than in healthy controls (p = 0.002). Healthy females had a significantly higher concentration of IL-12p40 than healthy males (p = 0.009). Female patients with schizophrenia had significantly higher concentrations of IL-12p40 than their male counterparts (p < 0.001), healthy females (p = 0.018), and healthy males (p < 0.001), respectively. Male patients with schizophrenia had significantly higher concentrations of IL-12p40 than healthy males (p = 0.023). The

  5. Inflammation and Elevation of Interleukin-12p40 in Patients with Schizophrenia

    PubMed Central

    Bedrossian, Nora; Haidar, Mariam; Fares, Jawad; Kobeissy, Firas H.; Fares, Youssef

    2016-01-01

    Schizophrenia is a serious mental illness with chronic symptoms and significant impairment in psychosocial functioning, which suggests that it likely has neurodegenerative characteristics. Inflammatory markers such as pro-inflammatory cytokines are well-known etiological contributors for psychiatric disorders, including schizophrenia. Although, the role of inflammation in schizophrenia is becoming evident, the number of studies in this area is relatively scarce, especially in Lebanon, and increased procedural thoroughness is needed. Cytokines play a key role in the activation of the immune system and strongly influence neurotransmission. Previous investigation of plasma levels showed dysregulation of interleukin (IL)-12. However, genotypical variations of this interleukin have not been investigated for patients with schizophrenia yet. Thus, in this paper, we aimed to compute and assess IL-12p40 levels in the sera of individuals with schizophrenia from different provinces in Lebanon and compare it to controls. Healthy subjects comprised 60 individuals with a male/female (M/F) ratio of 31/29, whereas patients with schizophrenia consisted of 63 subjects with an M/F ratio of 30/33. The mean age for healthy controls was 30 years, whereas that for patients with schizophrenia was 35 years. A standardized enzyme-linked immunosorbent assay (ELISA) technique was used to measure the concentration of IL-12p40 in all collected sera (n = 123). The mean IL-12p40 levels in patients with schizophrenia were significantly higher than in healthy controls (p = 0.002). Healthy females had a significantly higher concentration of IL-12p40 than healthy males (p = 0.009). Female patients with schizophrenia had significantly higher concentrations of IL-12p40 than their male counterparts (p < 0.001), healthy females (p = 0.018), and healthy males (p < 0.001), respectively. Male patients with schizophrenia had significantly higher concentrations of IL-12p40 than healthy males (p = 0.023). The

  6. Essential Role of Interleukin-12/23p40 in the Development of Graft-versus-Host Disease in Mice.

    PubMed

    Wu, Yongxia; Bastian, David; Schutt, Steven; Nguyen, Hung; Fu, Jianing; Heinrichs, Jessica; Xia, Changqing; Yu, Xue-Zhong

    2015-07-01

    Graft-versus-host disease (GVHD), in both its acute (aGVHD) and chronic (cGVHD) forms, remains a major obstacle impeding successful allogeneic hematopoietic stem cell transplantation (allo-HSCT). T cells, in particular pathogenic T helper (Th) 1 and Th17 subsets, are a driving force for the induction of GVHD. IL-12 and IL-23 cytokines share a common p40 subunit and play a critical role in driving Th1 differentiation and in stabilizing the Th17 phenotype, respectively. In our current study, we hypothesized that p40 is an essential cytokine in the development of GVHD. By using p40-deficient mice, we found that both donor- and host-derived p40 contribute to the development of aGVHD. Neutralization of p40 with an anti-p40 mAb inhibited Th1- and Th17-polarization in vitro. Furthermore, anti-p40 treatment reduced aGVHD severity while preserving the graft-versus-leukemia (GVL) activity. Alleviation of aGVHD was associated with an increase of Th2 differentiation and a decrease of Th1 and Th17 effector T cells in the GVHD target organs. In addition, anti-p40 treatment attenuated the severity of sclerodermatous cGVHD. These results provide a strong rationale that blockade of p40 may represent a promising therapeutic strategy in preventing and treating aGVHD and cGVHD while sparing the GVL effect after allo-HSCT. PMID:25846718

  7. The Structure of Interleukin-23 Reveals in the Molecular Basis of P40 Subunit Sharing With Interleukin-12

    SciTech Connect

    Lupardus, P.J.; Garcia, K.C.

    2009-05-19

    Interleukin-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an {alpha}-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3{angstrom} crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an Arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of the pocket on p40. This 'Arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central Arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pair-wise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.

  8. Differential expression of interleukin-12 p35 and p40 subunits in response to Aeromonas hydrophila and Aquareovirus infection in grass carp, Ctenopharyngodon idella.

    PubMed

    Pandit, N P; Shen, Y B; Xu, X Y; Yu, H Y; Wang, W J; Wang, R Q; Xuan, Y F; Li, J L

    2015-01-01

    The grass carp (Ctenopharyngodon idella) aquaculture industry in Asia is prone to bacterial and viral hemorrhagic diseases. Effective adjuvants for vaccine formulation are the need of the hour for control of these diseases and long-term sustainability of grass carp farming. In this study, the involvement of interleukin-12 (IL-12) from grass carp (gcIL‑12) in anti-bacterial and anti-viral immune responses was demonstrated via expression profiles of gcIL-12 subunits in immune tissues of the fish, following infection by Aeromonas hydrophila and Aquareovirus. Additionally, cDNA of the gcIL-12 subunits, p35 and p40 was cloned and characterized. We found that most of the structurally and functionally important features of vertebrate orthologues were conserved in gcIL-12 subunits, p35 and p40, with some features specific to grass carp. High levels of gcIL-12 p35 expression in the brain and gills suggest that IL-12 plays an important role in neural and immune systems. High expression levels in the heart, blood, and immune-related tissues suggest an important role in circulation and the immune system as well. Infections by both, A. hydrophila and Aquareovirus stimulated the mRNA expression of gcIL-12 subunits, p35 and p40 in most immune tissues. Significant upregulation or downregulation of gcIL-12 subunits, p35 and p40 by bacterial and viral infection confirms their potential role in anti-bacterial and anti-viral immune responses in fish. PMID:25730056

  9. Interferon Consensus Sequence Binding Protein–deficient Mice Display Impaired Resistance to Intracellular Infection Due to a Primary Defect in Interleukin 12 p40 Induction

    PubMed Central

    Scharton-Kersten, Tanya; Contursi, Cristina; Masumi, Atsuko; Sher, Alan; Ozato, Keiko

    1997-01-01

    Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family of transcription proteins, were infected with the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-γ and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP−/− animals. In related experiments, macrophages from uninfected ICSBP−/− mice were shown to display a selective impairment in the mRNA expression of IL-12 p40 but not IL-1α, IL-1β, IL-1Ra, IL-6, IL-10, or TNF-α in response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-γ. We hypothesize that the impaired synthesis of IL-12 p40 in ICSBP−/− animals is the primary lesion responsible for the loss in resistance to T. gondii because IFN-γ–induced parasite killing was unimpaired in vitro and, more importantly, administration of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-γ–dependent host resistance. PMID:9348310

  10. The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus

    PubMed Central

    Huante-Mendoza, Alejandro; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J.; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2015-01-01

    Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus. PMID:26200352

  11. Interleukin-12 gene-expression of macrophages is regulated by nitric oxide.

    PubMed

    Rothe, H; Hartmann, B; Geerlings, P; Kolb, H

    1996-07-01

    Interleukin-12 is a heterodimeric cytokine, mainly produced by macrophages. In our present study we demonstrate that interleukin-12 expression is regulated by nitric oxide. Incubation of the macrophage cell line IC 21 with interferon-gamma gave rise to both interleukin-12 p40 mRNA and nitric oxide production. The concurrent addition of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine inhibited nitrite production and in parallel completely suppressed interleukin-12 p40 mRNA formation. This indicated that endogenous nitric oxide synthase activity was required for IL-12 p40 gene expression. Exposure of the cells towards the nitric oxide generating compounds nitroprusside or S-nitroso-N-acetyl-penicillamine induced interleukin-12 p40 mRNA. Maximal mRNA levels were induced with nitric oxide donors at 1 microM concentration. We conclude that nitric oxide may exert an autoregulatory and paracrine control of interleukin-12 gene expression. PMID:8694804

  12. A protease-activated receptor 2 agonist (AC-264613) suppresses interferon regulatory factor 5 and decreases interleukin-12p40 production by lipopolysaccharide-stimulated macrophages: Role of p53.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-06-01

    The transcription factor interferon regulatory factor 5 (IRF5) has a key role in the production of interleukin (IL)-12 by macrophages. IRF5 is also a central mediator of toll-like receptor signaling and is a direct target of p53. Activation of protease-activated receptor 2 (PAR-2) upregulates p53 and suppresses apoptosis. This study investigated the influence of human neutrophil elastase (HNE) and PAR-2 agonists on expression of IRF5 and IL-12p40 by macrophages stimulated with lipopolysaccharide. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophages showed upregulation of IRF5 expression, while HNE reduced expression of p53 and IRF5 in a concentration-dependent manner. HNE also caused a concentration-dependent decrease of IRF5 in macrophages transfected with small interfering RNA to silence p53, while silencing of β-arrestin 2 blunted the reduction of p53 or IRF5 by HNE. Incubation of macrophages with a PAR-2 agonist, AC-264613, caused a decrease of IRF5 expression and also significantly reduced p53 protein expression. HNE upregulated the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and caused transactivation of TLR4, while AC-264613 did not promote TLR4 transactivation. In conclusion, the PAR-2 agonist AC-264613 attenuated IRF5-associated IL-12p40 production by macrophages. PMID:26833899

  13. Interleukin-12 in infectious diseases.

    PubMed Central

    Romani, L; Puccetti, P; Bistoni, F

    1997-01-01

    Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that is crucially involved in a wide range of infectious diseases. In several experimental models of bacterial, parasitic, viral, and fungal infection, endogenous IL-12 is required for early control of infection and for generation and perhaps maintenance of acquired protective immunity, directed by T helper type 1 (Th1) cells and mediated by phagocytes. Although the relative roles of IL-12 and gamma interferon in Th1-cell priming may be to a significant extent pathogen dependent, common to most infections is that IL-12 regulates the magnitude of the gamma interferon response at the initiation of infection, thus potentiating natural resistance, favoring Th1-cell development; and inhibiting Th2 responses. Treatment of animals with IL-12, either alone or as a vaccine adjuvant, has been shown to prevent disease by many of the same infectious agents, by stimulating innate resistance or promoting specific reactivity. Although IL-12 may enhance protective memory responses in vaccination or in combination with antimicrobial chemotherapy, it is yet unclear whether exogenous IL-12 can alter established responses in humans. Continued investigation into the possible application of IL-12 therapy to human infections is warranted by the role of the cytokine in inflammation, immunopathology, and autoimmunity. PMID:9336665

  14. Interleukin 12 at the site of disease in tuberculosis.

    PubMed Central

    Zhang, M; Gately, M K; Wang, E; Gong, J; Wolf, S F; Lu, S; Modlin, R L; Barnes, P F

    1994-01-01

    Interleukin 12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent immunologic effects in vitro. We used tuberculous pleuritis as a model to study the immunoregulatory potential of IL-12 in vivo at the site of human infectious disease. Messenger RNAs for p40 and p35 were detected in pleural fluid from six of six patients by reverse-transcription polymerase chain reaction. By using an ELISA that detected both free p40 and heterodimeric IL-12, we found that mean concentrations were 585 +/- 89 pg/ml in pleural fluid of patients with tuberculous pleuritis, which were significantly higher than those in serum of the same patients (54 +/- 36 pg/ml), or in malignant pleural effusions (123 +/- 35 pg/ml). By using an ELISA specific for heterodimeric IL-12, we found that mean concentrations in pleural fluid of patients with tuberculous pleuritis were 165 +/- 28 pg/ml and undetectable in serum of the same patients, or in malignant pleural effusions. Bioactive IL-12 was detectable in five of five supernatants of pleural fluid cells stimulated with Mycobacterium tuberculosis. Addition of anti-IL-12 antibodies suppressed proliferative responses of pleural fluid cells to M. tuberculosis by 36 +/- 7%. These data indicate that IL-12 may play a role in the human immune response to infectious agents in vivo. We hypothesize that IL-12 contributes to the antimycobacterial immune response by enhancing production of interferon-gamma, facilitating development of Th1 cells and augmenting cytotoxicity of antigen-specific T cells and natural killer cells. Images PMID:7909320

  15. Interleukin-12 is not essential for silicosis in mice

    PubMed Central

    Davis, Gerald S; Pfeiffer, Linda M; Hemenway, David R; Rincon, Mercedes

    2006-01-01

    Background Silicosis features foci of inflammation where macrophages and lymphocytes precede and accompany fibroblast proliferation, alveolar epithelial hyperplasia, and increased deposition of connective tissue matrix material. In the mouse following silica inhalation there is recruitment of natural killer-, B-, and CD4+ and CD8+ lymphocytes to the alveolar spaces, enlargement of bronchial-associated lymphoid tissues (BALT), and aggregation of lymphocytes surrounding small airways and blood vessels. A substantial fraction of the recruited lung lymphocytes produce interferon-γ (IFN-γ), and IFN-γ gene-deleted mice develop less silicosis than wild-type mice. Interleukin-12 (IL-12) is an important pathway for driving the adaptive immune response towards a TH1-like phenotype. We hypothesized that IL-12 might stimulate lymphocyte activation and the up-regulation of IFN-γ, and consequently be an essential mediator for silicosis. Results C57Bl/6 wild-type (WT) and IL-12 deficient (IL-12 KO) mice were exposed to sham-air or crystobalite silica (61 mg/m3) by inhalation for 5 hours/day for 12 days and then studied from 1 to 112 days after exposure. Mice exposed to sham-air had normal lung histology at all time points. WT mice exposed to titanium dioxide (72 mg/m3) showed pulmonary macrophage recruitment but no increase in lung collagen. Both WT and IL-12 KO mice exposed to silica showed similar progressive lung pathology, increased wet lung weight and increased total lung collagen (hydroxyproline). IL-12 p35 mRNA was not increased in either strain after silica exposure; IL-12 p40 mRNA was up-regulated after silica in WT mice and constitutively absent in the IL-12 KO mice. IL-18 mRNA was not increased after silica exposure. The expression of IL-15 (an important driver for innate immunity, Natural Killer cell activation, and IFN-γ production) was abundant in air-exposed mice and was increased slightly in the lungs of mice with silicosis. Conclusion The axis of IL-12

  16. Intrathecal production of interleukin-12 and gamma interferon in patients with bacterial meningitis.

    PubMed Central

    Kornelisse, R F; Hack, C E; Savelkoul, H F; van der Pouw Kraan, T C; Hop, W C; van Mierlo, G; Suur, M H; Neijens, H J; de Groot, R

    1997-01-01

    To assess the role of interleukin-12 (IL-12) and gamma interferon (IFN-gamma) in children with bacterial meningitis, bioactive IL-12 (p70) and the inactive subunit p40 and IFN-gamma were measured in serum and cerebrospinal fluid (CSF) from 35 children with bacterial meningitis and 10 control subjects. The production of IFN-gamma is induced by IL-12 with tumor necrosis factor alpha (TNF-alpha) as a costimulator and inhibited by IL-10. CSF concentrations of IL-12 p40 as well as those of IFN-gamma were markedly elevated, whereas IL-12 p70 was hardly detectable. Detectable CSF levels of IFN-gamma correlated positively with IL-12 p40 (r = 0.40, P = 0.02) and TNF-alpha (r = 0.46, P = 0.04) but not with IL-6, IL-8, or IL-10. In contrast to CSF levels of TNF-alpha, IL-12, and IL-10, those of IFN-gamma were significantly higher in patients with pneumococcal meningitis than in children with meningitis caused by Haemophilus influenzae and Neisseria meningitidis, presumably because of a high CSF TNF-alpha/IL-10 ratio in the former. We suggest that IL-12- and TNF-alpha-induced IFN-gamma production may contribute to the natural immunity against microorganisms in the CSF compartment during the acute phase of bacterial meningitis. PMID:9038291

  17. 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

  18. Anti-nociceptive effect of IL-12p40 in a rat model of neuropathic pain.

    PubMed

    Chen, I-Fang; Khan, Junad; Noma, Noboru; Hadlaq, Emad; Teich, Sorin; Benoliel, Rafael; Eliav, Eli

    2013-06-01

    IL-12p70 is a proinflammatory cytokine secreted by dendritic cells, monocytes and macrophages. It plays a crucial role in cell-mediated immunity by inducing proliferation of T cell and natural killer cells, and enhancing their cytotoxic activity. In adaptive immune response, it acts on naive T cells to differentiate into Th1-type cells. It is composed of two subunits, p35 and p40. The latter can be secreted in the form of monodimer or heterodimer, which is also referred as IL-12p80. Recently IL-12p70 has been proven to locally provoke nociceptive effect in naïve rats. This study investigated pain response following systemic administration of IL-12p70 and IL-12p40 homodimer in chronic neuropathic pain model, induced by chronic constriction injury. The doses tested were IL-12p40 homodimer or IL12p70 at 15, 150 and 1500ng/kg, respectively. Pain was assessed at 1, 4, 7 and 24h after injection, in the form of tactile allodynia and mechanical hyperalgesia. The side effect of sensory motor disability was measured by rotarod performance. By all behavioral measures, IL-12p70 of any dosage, at any time point, had no significant effect on tactile allodynia and mechanical hyperalgesia. A high dose of IL-12p40 homodimer induced significant analgesic effect by the measure of hind paw tactile allodynia from 1h to 4h after injection. Medium and low doses of IL-12p40 homodimer exerted their analgesic effect 4h post injection. Mechanical hyperalgesia, following high and medium doses of IL-12p40 administration, was significantly reduced at 4h after application. Also, no significant sensory motor dysfunction was detected for all dosage for both homodimers. These findings suggest that systemic application of IL-12p40 homodimer induces time-dependent analgesia to mechanical stimulation in rats exposed to neuropathic pain. PMID:23597590

  19. Interleukin-12 inhibits hepatitis B virus replication in transgenic mice.

    PubMed Central

    Cavanaugh, V J; Guidotti, L G; Chisari, F V

    1997-01-01

    Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection. PMID:9060687

  20. Development of Interleukin-12-Producing Capacity throughout Childhood

    PubMed Central

    Upham, John W.; Lee, Peter T.; Holt, Barbara J.; Heaton, Tricia; Prescott, Susan L.; Sharp, Mary J.; Sly, Peter D.; Holt, Patrick G.

    2002-01-01

    Increasing evidence indicates that the capacity to induce protective Th1 immune responses is impaired in early childhood, an observation that can be partially attributed to deficiencies in antigen-presenting-cell function. Synthesis of interleukin 12 (IL-12), a key Th1-trophic cytokine, is markedly reduced in the neonatal period, though there is a paucity of knowledge concerning the ontogeny of IL-12-synthetic capacity throughout the childhood years. Hence, we examined the production of bioactive IL-12 p70 by circulating mononuclear cells in a population of healthy individuals. As expected, the capacity to synthesize IL-12 p70 in response to either lipopolysaccharide or heat-killed Staphylococcus aureus was markedly impaired at birth, even after priming of cells with gamma interferon. Surprisingly however, IL-12 p70 synthesis by peripheral blood mononuclear cells from both 5- and 12-year-old children was still substantially below that seen in adults, and this did not appear to be related to excessive production of IL-10. In contrast, dendritic cells from adults and neonates, derived from monocytes with granulocyte-macrophage colony-stimulating factor and IL-4, synthesized equivalent amounts of IL-12 p70 in response to microbial stimulation. This indicates that the impaired capacity for IL-12 synthesis in childhood is not an intrinsic property of circulating mononuclear cells but rather can be readily overcome in response to appropriate maturational stimuli. Because IL-12 arose predominantly from circulating HLA-DR+ cells that lacked B-cell- and monocyte-specific markers, we propose that the slow maturation of IL-12-synthetic capacity in the childhood years can be attributed to deficiencies in the number and/or function of dendritic cells. PMID:12438328

  1. Curtiss P-40K Warhawk

    NASA Technical Reports Server (NTRS)

    1943-01-01

    Curtiss P-40K Warhawk: The Curtiss P-40K Warhawk was an improved version of the P-40E. This Warhawk was used by the NACA at Langley from July of 1942 until October of 1944. The P-40 gained most of its fame as the mount of the American Volunteer Group (AVG) 'Flying Tigers.' But shark mouthed P-40s were also flown by the Royal Air Force in North Africa.

  2. Expression of Epstein–Barr virus-induced gene 3 and other interleukin-12-related molecules by human intestinal epithelium

    PubMed Central

    Maaser, Christian; Egan, Laurence J; Birkenbach, Mark P; Eckmann, Lars; Kagnoff, Martin F

    2004-01-01

    Antigen-presenting cells, including dendritic cells, monocytes and macrophages, produce members of the interleukin-12 (IL-12) family that are important in initiating and maintaining cell-mediated immune responses. These include IL-12p35 and p19 that dimerize with IL-12p40 to form IL-12 (also termed IL-12p75) and IL-23, respectively, and Epstein–Barr virus-induced gene 3 (EBI3) protein (a protein related to IL-12p40), that forms a dimer with p28, termed IL-27. Intestinal epithelial cells, which are the initial site of contact between the host and enteric pathogens, can act as antigen-presenting cells, and are known to express mediators important in inflammatory and immune responses. In the current studies, we hypothesized that intestinal epithelial cells express members of the IL-12 family, which can function as an early signalling system important in mucosal immunity. Using in vitro and in vivo model systems of human intestinal epithelium, we demonstrate the regulated expression of EBI3, IL-12p35 and p19 by human intestinal epithelial cells. However, intestinal epithelial cells do not coexpress IL-12p40 or p28 that are required to generate heterodimeric IL-12p75, IL-23 and IL-27. To the extent that IL-12p35, p19 and EBI3 cannot form IL-12p75, IL-23 or IL-27 heterodimers in intestinal epithelial cells, these data suggest that those cells may express other, currently unknown, molecules that can associate with EBI3, IL-12p35 and/or p19 or, alternatively, intestinal epithelial cells may release IL-12-related molecules that by themselves, or in combination with other molecules in the mucosal microenvironment, mediate biological activities. PMID:15196212

  3. Cutaneous leukocytoclastic vasculitis in a child with interleukin-12 receptor beta-1 deficiency.

    PubMed

    Kutukculer, Necil; Genel, Ferah; Aksu, Guzide; Karapinar, Bulent; Ozturk, Can; Cavusoglu, Cengiz; Casanova, Jean-Laurent; Fieschi, Claire

    2006-03-01

    We report a patient with complete interleukin-12 receptor beta-1 deficiency associated with cutaneous leukocytoclastic vasculitis. The patient experienced Bacille Calmette Guérin, Mycobacterium chelonae, and Salmonella enteritidis infection. Vasculitis affecting both small arteries and postcapillary venules due to deposition of immune complexes was probably caused by S. enteritidis and/or M. chelonae infection. PMID:16615980

  4. Curtiss P-40E Warhawk

    NASA Technical Reports Server (NTRS)

    1942-01-01

    Curtiss P-40E Warhawk: The Curtiss P-40E Warhawk was powered by a liquid-cooled Allison V-1710 engine. Used for testing modifications to the tail, this Warhawk was used by the NACA at Langley from March to July 1942.

  5. Role of interleukin-12 in the regulation of CD4+ T cell apoptosis in a mouse model of asthma

    PubMed Central

    KODAMA, T; KURIBAYASHI, K; NAKAMURA, H; FUJITA, M; FUJITA, T; TAKEDA, K; DAKHAMA, A; GELFAND, E W; MATSUYAMA, T; KITADA, O

    2003-01-01

    Allergic asthma, a chronic inflammatory disease of the airways, is characterized by the presence of T helper 2 cells and eosinophils in sputum, bronchoalveolar lavage, and mucosal biopsy specimens. Although the T helper 1-promoting cytokine, interleukin-12, is capable of inhibiting the T helper 2-driven asthma symptoms and bronchial responsiveness, the specific mechanisms underlying these interleukin-12 actions are unclear. The anti-allergic response to interleukin-12 is only partially dependent on interferon-γ, which induces apoptosis by enhancing expression of Fas antigen. We therefore investigated in vivo whether the anti-allergic action of interleukin-12 is mediated through induction of apoptosis. C57BL/6 mice immunized to ovalbumin by intraperitoneal injection were challenged three times with an ovalbumin aerosol every second day for 7 days. Recombinant interleukin-12 was administered intravenously after the final challenge. After the last ovalbumin challenge, mice were examined for effects of interleukin-12 on inflammatory cell infiltration and apoptosis in the lung as detected by terminal deoxynucleotidyl transferase-mediated deoxyribonucleoside triphosphate nick end-labelling. Administration of interleukin-12 reduced ovalbumin-induced pulmonary eosinophilia (P < 0·01) and CD4+ T cell infiltration (P < 0·01). Moreover, treatment with interleukin-12 shortly after ovalbumin inhalation resulted in both increased interferon-γ production (P < 0·01) and enhanced apoptosis of CD4+ T cells in allergic airway infiltrates (P < 0·05). These results suggest that the beneficial effects of interleukin-12 in asthma may include enhancement of apoptosis of CD4+ T cells in airways. PMID:12562378

  6. Identification and functional characterization of multiple interleukin 12 in amberjack (Seriola dumerili).

    PubMed

    Matsumoto, Megumi; Hayashi, Kazuma; Suetake, Hiroaki; Yamamoto, Atsushi; Araki, Kyosuke

    2016-08-01

    Interleukin (IL) -12 is a heterodimeric cytokine mainly produced by monocytes, macrophages, and dendritic cells in mammals. IL-12p70 composed of IL-12p35 and IL-12p40, is known to play a crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFN-γ production. Although two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) have been identified in several fish species, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we cloned two types of IL-12p35 and three types of IL-12p40 genes in amberjack and yellowtail, and analyzed their expressions in response to stimulation with Nocardia seriolae in amberjack. As a result, four types of IL-12 (IL-12p35a, p35b, p40a and p40b) and IFN-γ mRNA were increased by live-N. seriolae stimulation but not by formalin-killed N. seriolae, suggesting that four types of IL-12 (p35, p35b, p40a and p40c) participate in promoting CMI. Subsequently, we produced six types of recombinant IL-12p70 (rIL12p70) protein in insect cells. Head kidney leukocytes were cultured with formalin-killed N. seriolae and six types of rIL-12p70 to elucidate the role of amberjack IL-12p70 in induction of CMI. After stimulation, IFN-γ expression was elevated whereas IL-10 expression was suppressed in Head kidney leukocytes stimulated with four types of rIL-12 (p40a/p35a, p40c/p35a, p40a/p35b, p40a/p35b). On the other hand, two types of rIL-12 (p40b/p35a, p40b/p35b) only elicited down regulation of IL-10 expression. These results indicate that all amberjack IL-12p70 isoforms are involved in Th1 -differentiation and promotion of CMI with different manners. Fish IL-12 has a potential for the promising vaccine adjuvant. PMID:27238429

  7. Lecithin retinol acyltransferase forms functional homodimers.

    PubMed

    Jahng, Wan Jin; Cheung, Eric; Rando, Robert R

    2002-05-21

    Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation. PMID:12009892

  8. 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. PMID:25796985

  9. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells

    PubMed Central

    Chiaruttini, Giulia; Piperno, Giulia M.; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A.; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-01-01

    Summary Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  10. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells.

    PubMed

    Chiaruttini, Giulia; Piperno, Giulia M; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-03-22

    Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  11. Myxoma virus expressing human interleukin-12 does not induce myxomatosis in European rabbits.

    PubMed

    Stanford, Marianne M; Barrett, John W; Gilbert, Philippe-Alexandre; Bankert, Richard; McFadden, Grant

    2007-11-01

    Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressing the complete repertoire of MV proteins. Thus, vMyxIL-12 exhibits promise as an oncolytic candidate and is safe in all known vertebrate hosts, including lagomorphs. PMID:17728229

  12. Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells.

    PubMed Central

    Matsumoto, H; Suzuki, K; Tsuyuguchi, K; Tanaka, E; Amitani, R; Maeda, A; Yamamoto, K; Sasada, M; Kuze, F

    1997-01-01

    Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens. In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation. A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline. Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold. In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression. These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay. Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression. Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression. However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta. We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma. PMID:9353012

  13. (p40)2-Fc reduces immune-inflammatory response through the activation of T cells in collagen induced arthritis mice.

    PubMed

    Lee, Seon-Yeong; Lee, Seung Hoon; Park, Seong-Jeong; Kim, Doo-Jin; Kim, Eun-Kyung; Kim, Jae-Kyung; Yang, Se-Hwan; Park, Sung-Hwan; Sung, Young-Chul; Kim, Ho-Youn; Cho, Mi-La

    2016-08-01

    IL-12p40 homodimer, a natural antagonist of IL-12 and IL-23, performs an important role in the expression of proinflammatory cytokines that is essential for Th1 and Th17 immune responses. Here, we reveal the therapeutic and immunosuppressive effect of the IL-12p40 subunit ((p40)2-Fc) in an experimental autoimmune arthritis model. We hypothesized that (p40)2-Fc may reduce the inflammatory response and the activation of T cells. In this study, we intraperitoneally injected (p40)2-Fc into collagen induced arthritis (CIA) mice to identify whether (p40)2-Fc attenuates CIA severity. (p40)2-Fc reduced the development of CIA, joint inflammation and cartilage destruction. (p40)2-Fc also significantly decreased the concentration of serum immunoglobulin as well as the number of T cells and C II specific T cells. In addition, osteoclastogenesis in (p40)2-Fc treated mice was down-regulated compared to the mice treated with (p40)2-Fc control. We observed that (p40)2-Fc treatment alleviates arthritis in mice with CIA, reducing inflammation and osteoclast differentiation. These findings suggest that (p40)2-Fc can be a potential therapeutic approach for autoimmune arthritis. PMID:27229912

  14. Cancer Therapeutic Based on T Cell Receptors Designed to Regiospecifically Release Interleukin-12 | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Surgery Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize a potential cancer therapeutic based on T cells genetically engineered to express the human interleukin 12 (IL-12) cytokine only in the tumor environment.

  15. GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers.

    PubMed

    Liu, Ying; Zhang, Yue; Jiang, Jing; Lobie, Peter E; Paulmurugan, Ramasamy; Langenheim, John F; Chen, Wen Y; Zinn, Kurt R; Frank, Stuart J

    2016-05-01

    GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers. PMID:27003442

  16. Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12.

    PubMed

    Liu, Yingru; Egilmez, Nejat K; Russell, Michael W

    2013-12-01

    Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as resistant to most available antibiotics, yet it does not induce a state of specific protective immunity against reinfection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer microspheres significantly enhanced both Th1 and humoral immune responses in a mouse model of genital gonococcal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specific circulating immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local administration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against gonorrhea, with implications for the development of an effective vaccine. PMID:24048962

  17. Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis

    PubMed Central

    1995-01-01

    When administered at or near the initiation of experimental intracellular infection caused by Leishmania major, Toxoplasma gondii, or Cryptococcus neoformans, treatment with the immuno-regulatory cytokine interleukin 12 (IL-12), induces protective antimicrobial activity. In contrast, once infections are established, IL-12 exerts considerably less or no effect in the face of a suppressive Th2 cell- associated response (L. major) or rapidly progressive fatal infection (T. gondii). To test the efficacy of IL-12 in an established intracellular protozoal infection but under quite different immunologic conditions (Th1 cell response, acquired resistance), L. donovani- infected BALB/c mice were treated starting 2 wk after challenge coincident with the onset of the Th1 cell response. In this environment, 7 d of IL-12 treatment reduced liver parasite burdens by 47%, an effect comparable to that induced by exogenous interferon (IFN) gamma. The in vivo mechanism responsive to IL-12 was complex, and required both CD4+ and CD8+ T cells as well as natural killer cells and the action of multiple endogenous antileishmanial cytokines (IFN-gamma, IL-2, tumor necrosis factor alpha). Early treatment with IL-12 before the expression of the Th1 cell response was also effective and induced an accelerated, near-cure response via an IFN-gamma-dependent mechanism. These results extend the antimicrobial-inducing capacity of IL-12 beyond prophylaxis by indicating that IL-12 can exert clear-cut therapeutic activity in an established intracellular infection. PMID:7807019

  18. Interleukin-12 reverses the inhibitory impact of photodynamic therapy (PDT) on the murine contact hypersensitivity response

    NASA Astrophysics Data System (ADS)

    Simkin, Guillermo O.; Levy, Julia G.; Hunt, David W. C.

    1998-05-01

    Treatment of mice with certain photosensitizers combined with exposure to visible light limits the development of the immunologically-mediated contact hypersensitivity (CHS) response against topically-applied chemical haptens. Understanding of the inhibitory action of photosensitizers upon the CHS response is incomplete. Benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin), a photosensitizer with immunomodulatory activity, strongly depressed CHS responses to the hapten dinitrofluorobenzene (DNFB). However, if mice were administered 1 (mu) g of a recombinant preparation of the pro- inflammatory cytokine interleukin-12 (rIL-12), full-fledged CHS responses to DNFB ensued in animals treated with BPD-MA and light. In contrast, when rIL-12 was given in combination with an anti-IL-12 antibody the restorative effect of rIL-12 on the CHS response of PDT-treated mice was blocked. Evaluation of the cytokine status of spleen and draining lymph node cells showed for DNFB painted animals, that the release of the immunosuppressive cytokine IL-10 was increased by PDT and rIL-12 counter-acted the increase in IL-10 liberation associated with PDT. These studies indicate that IL-10 formation is upregulated and the availability of IL-12 may be limited in mice treated with PDT. These features may contribute to deficient CHS responses observed with PDT.

  19. Interleukin-12 preserves the cutaneous physical and immunological barrier after radiation exposure.

    PubMed

    Gerber, Scott A; Cummings, Ryan J; Judge, Jennifer L; Barlow, Margaret L; Nanduri, Julee; Johnson, Doug E Milano; Palis, James; Pentland, Alice P; Lord, Edith M; Ryan, Julie L

    2015-01-01

    The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation and dispersal of radiological material are legitimate threats. Such attacks could have devastating consequences to large populations, in the form of radiation injury to various human organ systems. One of these at risk organs is the cutaneous system, which forms both a physical and immunological barrier to the surrounding environment and is particularly sensitive to ionizing radiation. Therefore, increased efforts to develop medical countermeasures for treatment of the deleterious effects of cutaneous radiation exposure are essential. Interleukin-12 (IL-12) was shown to elicit protective effects against radiation injury on radiosensitive systems such as the bone marrow and gastrointestinal tract. In this article, we examined if IL-12 could protect the cutaneous system from a combined radiation injury in the form of sublethal total body irradiation and beta-radiation burn (β-burn) directly to the skin. Combined radiation injury resulted in a breakdown in skin integrity as measured by transepidermal water loss, size of β-burn lesion and an exacerbated loss of surveillant cutaneous dendritic cells. Interestingly, intradermal administration of IL-12 48 h postirradiation reduced transepidermal water loss and burn size, as well as retention of cutaneous dendritic cells. Our data identify IL-12 as a potential mitigator of radiation-induced skin injury and argue for the further development of this cytokine as a radiation countermeasure. PMID:25564716

  20. Augmented anti-tumor effect of dendritic cells genetically engineered by interleukin-12 plasmid DNA.

    PubMed

    Yoshida, Masataka; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2010-01-01

    The objective of this study was to genetically engineer dendritic cells (DC) for biological activation and evaluate their anti-tumor activity in a tumor-bearing mouse model. Mouse DC were incubated on the surface of culture dishes which had been coated with the complexes of a cationized dextran and luciferase plasmid DNA complexes plus a cell adhesion protein, Pronectin, for gene transfection (reverse transfection). When compared with the conventional transfection where DC were transfected in the medium containing the complexes, the level of gene expression by the reverse method was significantly higher and the time period of gene expression was prolonged. Following the reverse transfection of DC by a plasmid DNA of mouse interleukin-12 (mIL-12) complexed with the cationized dextran, the mIL-12 protein was secreted at higher amounts for a longer time period. When injected intratumorally into mice carrying a mass of B16 tumor cells, the DC genetically activated showed significant anti-tumor activity. PMID:20338099

  1. Interleukin-12 Preserves the Cutaneous Physical and Immunological Barrier after Radiation Exposure

    PubMed Central

    Gerber, Scott A.; Cummings, Ryan J.; Judge, Jennifer L.; Barlow, Margaret L.; Nanduri, Julee; Milano Johnson, Doug E.; Palis, James; Pentland, Alice P.; Lord, Edith M.; Ryan, Julie L.

    2015-01-01

    The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation and dispersal of radiological material are legitimate threats. Such attacks could have devastating consequences to large populations, in the form of radiation injury to various human organ systems. One of these at risk organs is the cutaneous system, which forms both a physical and immunological barrier to the surrounding environment and is particularly sensitive to ionizing radiation. Therefore, increased efforts to develop medical countermeasures for treatment of the deleterious effects of cutaneous radiation exposure are essential. Interleukin-12 (IL-12) was shown to elicit protective effects against radiation injury on radiosensitive systems such as the bone marrow and gastrointestinal tract. In this article, we examined if IL-12 could protect the cutaneous system from a combined radiation injury in the form of sublethal total body irradiation and beta-radiation burn (β-burn) directly to the skin. Combined radiation injury resulted in a breakdown in skin integrity as measured by transepidermal water loss, size of β-burn lesion and an exacerbated loss of surveillant cutaneous dendritic cells. Interestingly, intradermal administration of IL-12 48 h postirradiation reduced transepidermal water loss and burn size, as well as retention of cutaneous dendritic cells. Our data identify IL-12 as a potential mitigator of radiation-induced skin injury and argue for the further development of this cytokine as a radiation countermeasure. PMID:25564716

  2. Toxoplasma gondii Upregulates Interleukin-12 To Prevent Plasmodium berghei-Induced Experimental Cerebral Malaria

    PubMed Central

    Settles, Erik W.; Moser, Lindsey A.; Harris, Tajie H.

    2014-01-01

    A chronic infection with the parasite Toxoplasma gondii has previously been shown to protect mice against subsequent viral, bacterial, or protozoal infections. Here we have shown that a chronic T. gondii infection can prevent Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. Treatment with soluble T. gondii antigens (STAg) reduced parasite sequestration and T cell infiltration in the brains of P. berghei-infected mice. Administration of STAg also preserved blood-brain barrier function, reduced ECM symptoms, and significantly decreased mortality. STAg treatment 24 h post-P. berghei infection led to a rapid increase in serum levels of interleukin 12 (IL-12) and gamma interferon (IFN-γ). By 5 days after P. berghei infection, STAg-treated mice had reduced IFN-γ levels compared to those of mock-treated mice, suggesting that reductions in IFN-γ at the time of ECM onset protected against lethality. Using IL-10- and IL-12βR-deficient mice, we found that STAg-induced protection from ECM is IL-10 independent but IL-12 dependent. Treatment of P. berghei-infected mice with recombinant IL-12 significantly decreased parasitemia and mortality. These data suggest that IL-12, either induced by STAg or injected as a recombinant protein, mediates protection from ECM-associated pathology potentially through early induction of IFN-γ and reduction in parasitemia. These results highlight the importance of early IL-12 induction in protection against ECM. PMID:24396042

  3. Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model

    PubMed Central

    2013-01-01

    Background Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12. Methods To test the inducibility of the p21 promoter, three reporter gene experimental models with green fluorescent protein (GFP) under the control of p21 promoter were established by gene electrotransfer of plasmid DNA: stably transfected cells, stably transfected tumors, and transiently transfected muscles. Induction of reporter gene expression after irradiation was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope in vivo. The antitumor effect of the plasmid encoding the p21 promoter driven interleukin 12 after radio-gene therapy was determined by tumor growth delay assay and by quantification of intratumoral and serum levels of interleukin 12 protein and intratumoral concentrations of interleukin 12 mRNA. Results Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter. Conclusions In this

  4. Anti-inflammatory cytokines in asthma and allergy: interleukin-10, interleukin-12, interferon-gamma.

    PubMed Central

    Chung, F

    2001-01-01

    Interleukin-10 (IL-10) is a cytokine derived from CD4+ T-helper type 2 (T(H2)) cells identified as a suppressor of cytokines from T-helper type 1(T(H1)) cells. Interleukin-12 (IL-12) is produced by B cells, macrophages and dendritic cells, and primarily regulates T(H1) cell differentiation, while suppressing the expansion of T(H2) cell clones. Interferon-gamma (IFN-gamma) is a product of T(H1) cells and exerts inhibitory effects on T(H2) cell differentiation. These cytokines have been implicated in the pathogenesis of asthma and allergies. In this context, IL-12 and IFN-gamma production in asthma have been found to be decreased, and this may reduce their capacity to inhibit IgE synthesis and allergic inflammation. IL-10 is a potent inhibitor of monocyte/macrophage function, suppressing the production of many pro-inflammatory cytokines. A relative underproduction of IL-10 from alveolar macrophages of atopic asthmatics has been reported. Therapeutic modulation of T(H1)/T(H2) imbalance in asthma and allergy by mycobacterial vaccine, specific immunotherapy and cytoline-guanosine dinucleotide motif may lead to increases in IL-12 and IFN-gamma production. Stimulation of IL-10 production by antigen-specific T-cells during immunotherapy may lead to anergy through inhibition of CD28-costimulatory molecule signalling by IL-10s anti-inflammatory effect on basophils, mast cells and eosinophils. PMID:11405550

  5. Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

    PubMed Central

    Cutrera, Jeffry; King, Glenn; Jones, Pamela; Kicenuik, Kristin; Gumpel, Elias; Xia, Xueqing; Li, Shulin

    2015-01-01

    Electroporation improves the anti-tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation-mediated chemotherapeutics with interleukin 12 (IL-12) plasmid DNA produces a strong yet safe anti-tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL-12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL-12 gene therapy for long-term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response-based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti-tumour response for successfully affecting not only the treated tumours, but also non-treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens. PMID:25628149

  6. Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

    PubMed Central

    Crettaz, Julien; Otano, Itziar; Ochoa, Laura; Benito, Alberto; Paneda, Astrid; Aurrekoetxea, Igor; Berraondo, Pedro; Rodríguez-Madoz, Juan Roberto; Astudillo, Aurora; Kreppel, Florian; Kochanek, Stefan; Ruiz, Juan; Menne, Stephan; Prieto, Jesus; Gonzalez-Aseguinolaza, Gloria

    2009-01-01

    Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 1010 viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-γ) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-γ in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 1010 vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers. PMID:19116251

  7. Intratumoral administration of a recombinant canarypox virus expressing interleukin 12 in patients with metastatic melanoma.

    PubMed

    Triozzi, Pierre L; Strong, Theresa V; Bucy, R Pat; Allen, Karen O; Carlisle, Ronda R; Moore, Susan E; Lobuglio, Albert F; Conry, Robert M

    2005-01-01

    The aim of this study was to evaluate the tolerability and activity of intratumoral administered human interleukin 12 encoded by a vector derived from the canarypox virus (ALVAC-IL-12). Nine patients with surgically incurable metastatic melanoma who had subcutaneous nodules available for injection were enrolled. ALVAC-IL-12 was administered by intratumoral injection on days 1, 4, 8, and 11. Tumor nodules greater than 2 cm in diameter were injected with 2 x 10(6) median tissue culture infectious doses (TCID(50)), and smaller tumors were injected with 1 x 10(6) TCID(50). The total dose per patient per time point ranged from 1 x 10(6) to 4 x 10(6) TCID(50). Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever associated with chills, myalgia, and fatigue. No dose-limiting toxicities occurred. Increases in IL-12 mRNA, and also increases in interferon gamma mRNA, were observed in ALVAC-IL-12-injected tumors compared with saline-injected control tumors in four of the nine patients. ALVAC-IL-12-injected tumors were also characterized by T cell infiltration. Three patients demonstrated increases in serum IL-12 and in interferon gamma levels. All patients developed neutralizing IgG antibody to the canarypox vector. One patient manifested a complete response of injected subcutaneous metastases and uninjected in-transit metastases. The intratumoral injection of ALVAC-IL-12 at these dose levels and according to this schedule was well tolerated and resulted in measurable biologic response in patients with metastatic melanoma. PMID:15703492

  8. Neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis

    PubMed Central

    Vo, Jimmy LN; Yang, Lirong; Kurtz, Samantha L; Smith, Sean G; Koppolu, Bhanu prasanth; Ravindranathan, Sruthi; Zaharoff, David A

    2015-01-01

    Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to

  9. Folate-Modified Lipoplexes Delivering the Interleukin-12 Gene for Targeting Colon Cancer Immunogene Therapy.

    PubMed

    Luo, Min; Liang, Xiao; Luo, Shun-Tao; Wei, Xia-Wei; Liu, Ting; Ren, Jun; Ma, Cui-Cui; Yang, Yu-Han; Wang, Bi-Lan; Liu, Li; Song, Xiang-Rong; He, Zhi-Yao; Wei, Yu-Quan

    2015-11-01

    The incidence and mortality rate of colorectal cancer increase every year, making it a serious threat to human health. Targeted immunogene therapy is a novel method of treating this type of cancer. Colon cancer overexpresses folate receptor α (FRα) and folate-modified liposomes for colon cancer immunogene therapy may suppress tumor growth effectively. In this study, F-PLP/pIL12, an FRα-targeted lipoplex loading plasmid interleukin-12 (pIL12) was prepared and its physicochemical properties were characterized. Then the antitumor effect of F-PLP/pIL12 was studied in an in vivo model of CT-26 colon cancer. F-PLP/pIL12 was associated with about 56.6% tumor growth inhibition compared with the saline control. The production of malignant ascites was significantly less pronounced than in controls, and there were fewer tumor nodules and less overall tumor mass (P < 0.01). There was more IL12 expression and IFN-γ secretion in F-PLP/pIL12-treated tumor tissues, but there was less FRα expression. The antitumor mechanisms involved inducing tumor cell apoptosis, reducing microvessel density, and stimulating TNF-α secretion. In addition, there were fewer M2 macrophages in the tumor microenvironment of tissues stimulated with F-PLP/pIL12, which also activated the natural killer cells. H&E staining of vital organs suggested that F-PLP/pIL12 is safe for use in intraperitoneally administered cancer therapy. It was here concluded that F-PLP/plL12 may be a suitable targeting formulation for colon cancer immunogene therapy. PMID:26554159

  10. Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

    PubMed Central

    Kyei, Stephen K.; Ogbomo, Henry; Li, ShuShun; Timm-McCann, Martina; Xiang, Richard F.; Huston, Shaunna M.; Ganguly, Anutosh; Colarusso, Pina; Gill, M. John

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans. Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. PMID:27555306

  11. Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination with Plasmid Interleukin 12 and Impact of Intramuscular Electroporation for Delivery

    PubMed Central

    Kalams, Spyros A.; Parker, Scott D.; Elizaga, Marnie; Metch, Barbara; Edupuganti, Srilatha; Hural, John; De Rosa, Stephen; Carter, Donald K.; Rybczyk, Kyle; Frank, Ian; Fuchs, Jonathan; Koblin, Beryl; Kim, Denny H.; Joseph, Patrice; Keefer, Michael C.; Baden, Lindsey R.; Eldridge, John; Boyer, Jean; Sherwat, Adam; Cardinali, Massimo; Allen, Mary; Pensiero, Michael; Butler, Chris; Khan, Amir S.; Yan, Jian; Sardesai, Niranjan Y.; Kublin, James G.; Weiner, David B.

    2013-01-01

    Background. DNA vaccines have been very poorly immunogenic in humans but have been an effective priming modality in prime-boost regimens. Methods to increase the immunogenicity of DNA vaccines are needed. Methods. HIV Vaccine Trials Network (HVTN) studies 070 and 080 were multicenter, randomized, clinical trials. The human immunodeficiency virus type 1 (HIV-1) PENNVAX®-B DNA vaccine (PV) is a mixture of 3 expression plasmids encoding HIV-1 Clade B Env, Gag, and Pol. The interleukin 12 (IL-12) DNA plasmid expresses human IL-12 proteins p35 and p40. Study subjects were healthy HIV-1–uninfected adults 18–50 years old. Four intramuscular vaccinations were given in HVTN 070, and 3 intramuscular vaccinations were followed by electroporation in HVTN 080. Cellular immune responses were measured by intracellular cytokine staining after stimulation with HIV-1 peptide pools. Results. Vaccination was safe and well tolerated. Administration of PV plus IL-12 with electroporation had a significant dose-sparing effect and provided immunogenicity superior to that observed in the trial without electroporation, despite fewer vaccinations. A total of 71.4% of individuals vaccinated with PV plus IL-12 plasmid with electroporation developed either a CD4+ or CD8+ T-cell response after the second vaccination, and 88.9% developed a CD4+ or CD8+ T-cell response after the third vaccination. Conclusions. Use of electroporation after PV administration provided superior immunogenicity than delivery without electroporation. This study illustrates the power of combined DNA approaches to generate impressive immune responses in humans. PMID:23840043

  12. Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy

    PubMed Central

    Zhou, Yedi; Yoshida, Shigeo; Kubo, Yuki; Kobayashi, Yoshiyuki; Nakama, Takahito; Yamaguchi, Muneo; Ishikawa, Keijiro; Nakao, Shintaro; Ikeda, Yasuhiro; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization. PMID:27312090

  13. Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy.

    PubMed

    Zhou, Yedi; Yoshida, Shigeo; Kubo, Yuki; Kobayashi, Yoshiyuki; Nakama, Takahito; Yamaguchi, Muneo; Ishikawa, Keijiro; Nakao, Shintaro; Ikeda, Yasuhiro; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization. PMID:27312090

  14. Computational design and experimental verification of a symmetric protein homodimer.

    PubMed

    Mou, Yun; Huang, Po-Ssu; Hsu, Fang-Ciao; Huang, Shing-Jong; Mayo, Stephen L

    2015-08-25

    Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein-protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by β-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix-mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability. PMID:26269568

  15. Interleukin-12 suppresses immunoglobulin E production but enhances immunoglobulin G4 production by human peripheral blood mononuclear cells.

    PubMed Central

    de Boer, B A; Kruize, Y C; Rotmans, P J; Yazdanbakhsh, M

    1997-01-01

    The effect of interleukin-12 (IL-12) on human immunoglobulin G4 (IgG4) and IgE production was examined with cells derived from filarial patients and European controls. IL-12 inhibited IgE release but enhanced IgG4 production in cultures of peripheral blood mononuclear cells stimulated with anti-CD2 plus IL-2. When purified T- and B-cell cocultures were examined, IL-12 again markedly enhanced IgG4, whereas IgE production was no longer inhibited. PMID:9038328

  16. Prospects for use of interleukin-12 as a mucosal adjuvant for vaccination of humans to protect against respiratory pneumococcal infection.

    PubMed

    Wright, A K A; Briles, D E; Metzger, D W; Gordon, S B

    2008-09-01

    Mucosal vaccination against pneumococcal disease offers potential protection against otitis media, pneumonia and invasive disease, including providing herd benefit by reducing pathogen carriage. The major obstacle, however, remains the lack of a suitable adjuvant for use in humans. Animal models have demonstrated success of interleukin-12 (IL-12) as an adjuvant for mucosal vaccines using recombinant pneumococcal protein antigens. This review examines the biology of the IL-12 cytokine family, the toxicity of IL-12 in human studies and suggests approaches by which IL-12 could be developed as a mucosal adjuvant with pneumococcal protein based vaccines, for use in humans. PMID:18602438

  17. Observation of an E2 (Ubc9)-homodimer by crystallography.

    PubMed

    Alontaga, Aileen Y; Ambaye, Nigus D; Li, Yi-Jia; Vega, Ramir; Chen, Chih-Hong; Bzymek, Krzysztof P; Williams, John C; Hu, Weidong; Chen, Yuan

    2016-06-01

    Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2], [3], [4], [5], [6], [7], [8], [9], [10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10], [11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The

  18. A Model to Explain How the Bacille Calmette Guérin (BCG) Vaccine Drives Interleukin-12 Production in Neonates.

    PubMed

    Kativhu, Chido Loveness; Libraty, Daniel H

    2016-01-01

    The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins. PMID:27571272

  19. A Model to Explain How the Bacille Calmette Guérin (BCG) Vaccine Drives Interleukin-12 Production in Neonates

    PubMed Central

    Kativhu, Chido Loveness; Libraty, Daniel H.

    2016-01-01

    The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins. PMID:27571272

  20. 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. PMID:26801817

  1. Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

    PubMed

    Dunham, Stephen P; Flynn, J Norman; Rigby, Mark A; Macdonald, Julie; Bruce, Jennifer; Cannon, Celia; Golder, Matthew C; Hanlon, Linda; Harbour, David A; Mackay, Nancy A; Spibey, Norman; Jarrett, Oswald; Neil, James C

    2002-02-22

    A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection. PMID:11858854

  2. 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

  3. In vivo activity of plant-based interleukin-12 in the lung of Balb/c mouse

    PubMed Central

    2010-01-01

    Background In the last years, plants are being used for the production of a wide variety of biopharmaceuticals, including cytokines, and have the potential to serve as vehicles for mucosal administration of these molecules. We had previously reported the expression of a cytokine, interleukin-12 (IL-12), in transgenic tomato plants and had demonstrated that it retained its biologic activity in vitro. Findings In this work, we administered crude extracts of IL-12-containing tomato fruits to mice through the intratracheal route, measuring endogenous IL-12 and determining biologic activity by quantification of interferon-gamma (IFN-γ) in lungs and by histological analysis. IFN-γ expression in lungs, as well as histological analysis, indicate that tomato-expressed IL-12 retains its biologic activity and, most importantly, its effects are restricted to the site of administration. Conclusion Our results indicate that the functional activity of tomato-expressed IL-12 is comparable to that of commercial recombinant IL-12 when given via the mucosal route. This opens the possibility of using crude extracts prepared from tomatoes expressing IL-12 for certain immunotherapies. PMID:20507618

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

    PubMed

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

    2016-06-01

    Whereas cancer immunotherapy with cytokines in recent research was demonstrated effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system as well as of the Fas-mediated apoptosis by various mechanisms. We report the design and preliminary evaluation of the antitumor activity of a novel fusion protein-mIL-12/FasTI, consisting of mouse interleukin-12 and the transmembrane and intracellular domains of mouse Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase-3 activity of the clones when co-cultured with NK cells indicated that apoptosis was induced through Fas/FasL signaling pathway. The preliminary results suggest a synergized anticancer activity of the fusion protein. It may represent a promising therapeutic agent for cancer treatment. PMID:27081758

  5. Interleukin-12 and photocarcinogenesis

    SciTech Connect

    Katiyar, Santosh K.

    2007-11-01

    UV radiation induces immunosuppression and inflammatory responses, as well as oxidative stress and DNA damage, in skin cells and these various effects have been implicated in melanoma and nonmelanoma skin cancers, i.e., photocarcinogenesis. The cytokine interleukin (IL)-12 has been shown to possess potent antitumor activity in a wide variety of murine tumor models. In this review, we summarize the evidence that IL-12 plays a role in preventing photocarcinogenesis, and present a model of its possible mechanisms of action. Treatment of mice with IL-12 prevents UV-induced immunosuppression in a process mediated by repair of UV-induced damaged DNA. After exposure to the photocarcinogenesis protocol, the development of UV-induced tumors is more rapid and the tumor multiplicity and tumor size are significantly greater in IL-12-deficient or knockout (KO) mice than their wild-type counterparts. IL-12-deficiency in mice enhances the proliferation potential of tumor cells, and this may be one of the reasons for the rapid growth of the tumors and their greater size. The rate of malignant transformation of UV-induced papillomas to carcinomas also is higher in the IL-12 KO mice than in their wild-type counterparts in terms of carcinoma incidence and carcinoma multiplicity. UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and sunburn cells is lower, or repaired more rapidly, in wild-type mice than IL-12 KO mice. The IL-12-associated reduction in UV-specific CPDs is due to induction of DNA repair, and particularly enhancement of nucleotide-excision repair. We suggest that endogenous stimulation of IL-12 may protect the skin from UV-induced immunosuppression, DNA damage, and, ultimately, the risk of photocarcinogenesis. Taken together, this information suggests that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of photocarcinogenesis.

  6. Tumor Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma

    PubMed Central

    Zhang, Ling; Morgan, Richard A.; D.Beane, Joal; Zheng, Zhili; Dudley, Mark E.; Kassim, Sadik H.; Nahvi, Azam V.; Ngo, Lien T.; Sherry, Richard M.; Phan, Giao Q.; Hughes, Marybeth S.; Kammula, Udai S.; Feldman, Steven A.; Toomey, Mary Ann; Kerkar, Sid. P.; Restifo, Nicholas P.; Yang, James C.; Rosenberg, Steven A.

    2015-01-01

    Purpose Infusion of interleukin-12 (IL-12) can mediate anti-tumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the anti-tumor activity of adoptively transferred human tumor infiltrating lymphocytes (TIL) genetically engineered to secrete single-chain IL-12 selectively at the tumor site. Experimental design Thirty-three patients with metastatic melanoma were treated in a cell-dose escalation trial of autologous TIL transduced with a gene encoding a single chain IL-12 driven by a nuclear factor of activated T cells promoter (NFAT.IL12). No IL-2 was administered. Results The administration of 0.001-0.1 X 109 NFAT.IL12 transduced TIL to 17 patients resulted in a single objective response (5.9%). However, at doses between 0.3-3 X 109 cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short and the administered IL-12 producing cells rarely persisted at one month. Increasing cell doses were associated with high serum levels of IL-12 and gamma-interferon as well as clinical toxicities including liver dysfunction, high fevers and sporadic life threatening hemodynamic instability. Conclusions In this first-in-man trial, administration of TIL transduced with an inducible IL-12 gene mediated tumor responses in the absence of IL-2 administration using cell doses 10-100 fold lower than conventional TIL. However, due to toxicities, likely attributable to the secreted IL-12, further refinement will be necessary before this approach can be safely utilized in the treatment of cancer patients. PMID:25695689

  7. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms.

    PubMed Central

    Zhan, Y; Liu, Z; Cheers, C

    1996-01-01

    Both interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) are produced early in intracellular bacterial infection. Depletion of either IL-12 or TNF-alpha by a single injection of specific antibody 4 h before the injection of Brucella abortus 19 led to the exacerbation of infection 2 weeks later. Whereas the effect of IL-12 depletion on resistance was persistent and exacerbation was still significant 6 weeks later, the bacterial numbers in mice depleted of TNF-alpha were similar to the bacterial numbers in control infected mice by 6 weeks postinfection. Massive splenomegaly, which is often seen in 2-week Brucella-infected mice, was not observed in IL-12- or TNF-alpha-depleted mice. Both IL-12- and TNF-alpha-depleted mice showed reduced cell accumulation in the spleen compared with the massive cell accumulation in control infected mice. Granuloma formation in livers was much reduced in IL-12-depleted mice but not in TNF-alpha-depleted mice. Gamma interferon (IFN-gamma) production by cells from TNF-alpha-depleted mice was not significantly different from that of cells from control infected mice. In contrast, the production of IFN-gamma by both CD4+ and CD8+ T cells from IL-12-depleted mice was greatly reduced, compared with that from control infected mice. This effect was still observed when the antibody injection was delayed for up to 7 days postinfection, but injections of anti-IL-12 antibody into mice with established Brucella infection had no significant effect on IFN-gamma production by T cells. Taken together, these results suggested that IL-12 contributed to resistance mainly via an IFN-gamma-dependent pathway and had a profound effect on the induction of acquired cellular resistance. In contrast, TNF-alpha was involved in resistance possibly via direct action on effector cells and may not be essential for the induction of acquired cellular resistance. PMID:8698508

  8. Interleukin-12-secreting human papillomavirus type 16-transformed cells provide a potent cancer vaccine that generates E7-directed immunity.

    PubMed

    Hallez, S; Detremmerie, O; Giannouli, C; Thielemans, K; Gajewski, T F; Burny, A; Leo, O

    1999-05-01

    The development of a vaccine that would be capable of preventing or curing the (pre)cancerous lesions induced by genital oncogenic human papillomaviruses (HPVs) is the focus of much research. Many studies are presently evaluating vaccines based on the viral E6 and E7 oncoproteins, both of which are continually expressed by tumor cells. The success of a cancer vaccine relies, in large part, on the induction of a tumor-specific Th1-type immunity. In this study, we have evaluated the ability of B7-related and/or interleukin-12 (IL-12)-expressing, non-immunogenic murine HPV16-transformed BMK-16/myc cells, to achieve this goal. BMK-16/myc cells engineered to express surface B7-1 or B7-2 molecules remain tumorigenic in syngeneic BALB/c mice, suggesting that expression of these molecules alone is not sufficient to induce tumor regression. In contrast, mice injected with tumor cells engineered to secrete IL-12 remained tumor-free, demonstrating that IL-12 expression is sufficient to induce tumor rejection. IL-12-secreting BMK-16/myc cells were further shown to induce potent and specific long-term tumor resistance, even after irradiation. B7-1 was found to slightly but systematically improve anti-tumor immunity elicited by IL-12-secreting BMK-16/myc cells. Injection of irradiated B7-1/IL-12+ BMK-16/myc cells generates long-lasting, Th1-type, BMK-16/myc-directed immunity in tumor-resistant mice. These mice display a memory-type, E7-specific, cell-mediated immune response, which is potentially significant for clinical applications. PMID:10209958

  9. Combined gene therapy of endostatin and interleukin 12 with polyvinylpyrrolidone induces a potent antitumor effect on hepatoma

    PubMed Central

    Li, Pei-Yuan; Lin, Ju-Sheng; Feng, Zuo-Hua; He, Yu-Fei; Zhou, He-Jun; Ma, Xin; Cai, Xiao-Kun; Tian, De-An

    2004-01-01

    AIM: To study the antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) on mouse transplanted hepatoma. METHODS: Mouse endostatin eukaryotic plasmid (pSecES) with a mouse Igκ signal sequence inside and mouse IL-12 eukaryotic plasmid (pmIL-12) were transfected into BHK-21 cells respectively. Endostatin and IL-12 were assayed by ELISA from the supernant and used to culture endothelial cells and spleen lymphocytes individually. Proliferation of the latter was evaluated by MTT. H22 cells were inoculated into the leg muscle of mouse, which was injected intratumorally with pSecES/PVP, pmIL-12/PVP or pSecES + pmIL-12/PVP repeatedly. Tumor weight, serum endostatin and serum IL-12 were assayed. Tumor infiltrating lymphocytes, tumor microvessel density and apoptosis of tumor cells were also displayed by HE staining, CD31 staining and TUNEL. RESULTS: Endostatin and IL-12 were secreted after transfection, which could inhibit the proliferation of endothelial cells or promote the proliferation of spleen lymphocytes. Tumor growth was highly inhibited by 91.8% after injection of pSecES + pmIL-12/PVP accompanied by higher serum endostatin and IL-12, more infiltrating lymphocytes, fewer tumor vessels and more apoptosis cells compared with injection of pSecES/PVP, pmIL-12/PVP or vector/PVP. CONCLUSION: Mouse endostatin gene and IL-12 gene can be expressed after intratumoral injection with PVP. Angiogenesis of hepatoma can be inhibited synergisticly, lymphocytes can be activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma can be highly inhibited or eradiated. PMID:15259064

  10. Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

    PubMed Central

    Del Río, Laura; Buendía, Antonio J.; Sánchez, Joaquín; Gallego, María C.; Caro, María R.; Ortega, Nieves; Seva, Juan; Pallarés, Francisco J.; Cuello, Francisco; Salinas, Jesús

    2001-01-01

    A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminate Chlamydophila abortus infections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12−/− and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/− mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/− mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/− infected mice. Both WT and IL-12−/− mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge with C. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/− mice. The lack of IL-12 resulted in few infiltrating CD4+ T cells in the liver relative to the number in WT mice, although the number of CD8+ T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection. PMID:11447154

  11. Natural Killer Cells and Helicobacter pylori Infection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production

    PubMed Central

    Yun, Cheol H.; Lundgren, Anna; Azem, Josef; Sjöling, Åsa; Holmgren, Jan; Svennerholm, Ann-Mari; Lundin, B. Samuel

    2005-01-01

    Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection. PMID:15731046

  12. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells.

    PubMed

    Nishibayashi, Ryoichiro; Inoue, Ryo; Harada, Yuri; Watanabe, Takumi; Makioka, Yuko; Ushida, Kazunari

    2015-01-01

    Interleukin-12 (IL-12) is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB). Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR) 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells. PMID:26083838

  13. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells

    PubMed Central

    Nishibayashi, Ryoichiro; Inoue, Ryo; Harada, Yuri; Watanabe, Takumi; Makioka, Yuko; Ushida, Kazunari

    2015-01-01

    Interleukin-12 (IL-12) is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB). Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR) 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells. PMID:26083838

  14. Irsogladine Maleate Prevents Colitis in Interleukin-10 Gene-Deficient Mice by Reducing Interleukin-12 and -23 Production.

    PubMed

    Nakagawa, Tomoo; Katsuno, Tatsuro; Noguchi, Yoshiko; Mandai, Yasushi; Yoshihama, Sayuri; Saito, Keiko; Maruoka, Daisuke; Matsumura, Tomoaki; Arai, Makoto; Yokosuka, Osamu

    2015-01-01

    Irsogladine maleate (2,4-diamino-6-[2,5-dichlorophenyl]-s-triazine maleate; IM), an anti-peptic ulcer drug, may have a protective effect on the gastrointestinal mucosa. This study investigated the effects of IM on spontaneous colitis in interleukin-10 gene-deficient (IL-10(-/-)) mice. Five-week-old IL-10(-/-) mice were fed a control diet or one containing 100 ppm of IM for 10 weeks. Colonic tissues were evaluated morphologically and histologically. J774A.1 murine monocyte/macrophage cells were incubated with IM after lipopolysaccharide stimulation. mRNA expression was assessed by quantitative polymerase chain reaction (PCR) and protein concentration by enzyme-linked immunosorbent assay (ELISA). Colonic length, weight, and histological scores clearly demonstrated that spontaneous colitis was prevented in IL-10(-/-) mice fed a diet containing IM compared with those fed control diet. Levels of tumor necrosis factor-alpha (TNF-α) (-2.5-fold), IL-1β (-5.4), interferon-gamma (IFN-γ) (-4.5), IL-17 (-113.0), IL-12p35 (-21.0), IL-12p40 (-3.4), and IL-23p19 (-4.2) mRNA expression were significantly decreased in the colonic tissues of IM-treated animals, suggesting that oral treatment with IM suppressed the T-helper (Th)1/Th17 immune response in the colonic mucosa. An in vitro study using monocyte/macrophage cells to clarify the pharmacological action of IM indicated that IL-12p40 and IL-23p19 mRNA expression levels were dose-dependently decreased by IM treatment. ELISA showed that IL-12p40 and IL-23 protein secretion were significantly decreased by IM in a dose-dependent manner. Oral treatment with IM prevented spontaneous colitis in IL-10(-/-) mice by suppressing the colonic mucosal Th1/Th17 immune response through inhibition of IL-12 and -23 production in monocyte/macrophage cells. PMID:26521820

  15. A covalent homodimer probing early oligomers along amyloid aggregation.

    PubMed

    Halabelian, Levon; Relini, Annalisa; Barbiroli, Alberto; Penco, Amanda; Bolognesi, Martino; Ricagno, Stefano

    2015-01-01

    Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation. PMID:26420657

  16. A covalent homodimer probing early oligomers along amyloid aggregation

    PubMed Central

    Halabelian, Levon; Relini, Annalisa; Barbiroli, Alberto; Penco, Amanda; Bolognesi, Martino; Ricagno, Stefano

    2015-01-01

    Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation. PMID:26420657

  17. Vector description of electric and hydrophobic interactions in protein homodimers.

    PubMed

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2016-05-01

    This article describes the formation of homodimers from their constituting monomers, based on the rules set by a simple model of electric and hydrophobic interactions. These interactions are described in terms of the electric dipole moment (D) and hydrophobic moment vectors (H) of proteins. The distribution of angles formed by the two dipole moments of monomers constituting dimers were analysed, as well as the distribution of angles formed by the two hydrophobic moments. When these distributions were fitted to Gaussian curves, it was found that for biological dimers, the D vectors tend mostly to adopt a perpendicular arrangement with respect to each other, in which the constituting dipoles have the least interaction. A minor population tends towards an antiparallel arrangement implying maximum electric attraction. Also in biological dimers, the H vectors of most monomers tend to interact in such a way that the total hydrophobic moment of the dimer increases with respect to those of the monomers. This shows that hydrophobic moments have a tendency to align. In dimers originating in the crystallisation process, the distribution of angles formed by both hydrophobic and electric dipole moments appeared rather featureless, probably because of unspecific interactions in the crystallisation processes. The model does not describe direct interactions between H and D vectors although the distribution of angles formed by both vectors in dimers was analysed. It was found that in most cases these angles tended to be either small (both moments aligned parallel to each other) or large (antiparallel disposition). PMID:26658743

  18. Chemical shift imprint of intersubunit communication in a symmetric homodimer.

    PubMed

    Falk, Bradley T; Sapienza, Paul J; Lee, Andrew L

    2016-08-23

    Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig1") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers. PMID:27466406

  19. Deprotonated Dicarboxylic Acid Homodimers: Hydrogen Bonds and Atmospheric Implications.

    PubMed

    Hou, Gao-Lei; Valiev, Marat; Wang, Xue-Bin

    2016-04-21

    Dicarboxylic acids represent an important class of water-soluble organic compounds found in the atmosphere. In this work we are studying properties of dicarboxylic acid homodimer complexes (HO2C(CH2)nCO2(-)[HO2C(CH2)nCO2H], n = 0-12), as potentially important intermediates in aerosol formation processes. Our approach is based on experimental data from negative ion photoelectron spectra of the dimer complexes combined with updated measurements of the corresponding monomer species. These results are analyzed with quantum-mechanical calculations, which provide further information about equilibrium structures, thermochemical parameters associated with the complex formation, and evaporation rates. We find that upon formation of the dimer complexes the electron binding energies increase by 1.3-1.7 eV (30.0-39.2 kcal/mol), indicating increased stability of the dimerized complexes. Calculations indicate that these dimer complexes are characterized by the presence of strong intermolecular hydrogen bonds with high binding energies and are thermodynamically favorable to form with low evaporation rates. Comparison with the previously studied HSO4(-)[HO2C(CH2)2CO2H] complex (J. Phys. Chem. Lett. 2013, 4, 779-785) shows that HO2C(CH2)2CO2(-)[HO2C(CH2)2CO2H] has very similar thermochemical properties. These results imply that dicarboxylic acids not only can contribute to the heterogeneous complexes formation involving sulfuric acid and dicarboxylic acids but also can promote the formation of homogeneous complexes by involving dicarboxylic acids themselves. PMID:27032015

  20. Structural Heterogeneity in Transmembrane Amyloid Precursor Protein Homodimer Is a Consequence of Environmental Selection

    PubMed Central

    2015-01-01

    The 99 amino acid C-terminal fragment of amyloid precursor protein (C99), consisting of a single transmembrane (TM) helix, is known to form homodimers. Homodimers can be processed by γ-secretase to produce amyloid-β (Aβ) protein, which is implicated in Alzheimer’s disease (AD). While knowledge of the structure of C99 homodimers is of great importance, experimental NMR studies and simulations have produced varying structural models, including right-handed and left-handed coiled-coils. In order to investigate the structure of this critical protein complex, simulations of the C9915–55 homodimer in POPC membrane bilayer and DPC surfactant micelle environments were performed using a multiscale approach that blends atomistic and coarse-grained models. The C9915–55 homodimer adopts a dominant right-handed coiled-coil topology consisting of three characteristic structural states in a bilayer, only one of which is dominant in the micelle. Our structural study, which provides a self-consistent framework for understanding a number of experiments, shows that the energy landscape of the C99 homodimer supports a variety of slowly interconverting structural states. The relative importance of any given state can be modulated through environmental selection realized by altering the membrane or micelle characteristics. PMID:24926593

  1. Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

    PubMed Central

    Ghosh, Ritwik; Narasanna, Archana; Wang, Shizhen Emily; Liu, Shuying; Chakrabarty, Anindita; Balko, Justin M.; González-Angulo, Ana María; Mills, Gordon B.; Penuel, Elicia; Winslow, John; Sperinde, Jeff; Dua, Rajiv; Pidaparthi, Sailaja; Mukherjee, Ali; Leitzel, Kim; Kostler, Wolfgang J.; Lipton, Allan; Bates, Michael; Arteaga, Carlos L.

    2011-01-01

    In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2+ breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated non-transformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. As expected, trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of HER2-overexpressing patients. Together, our findings corroborate the hypothesis that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers reflects an inability to activate the PI3K/AKT pathway. One of the most important clinical implications of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab. PMID:21324925

  2. Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers.

    PubMed

    Ghosh, Ritwik; Narasanna, Archana; Wang, Shizhen Emily; Liu, Shuying; Chakrabarty, Anindita; Balko, Justin M; González-Angulo, Ana María; Mills, Gordon B; Penuel, Elicia; Winslow, John; Sperinde, Jeff; Dua, Rajiv; Pidaparthi, Sailaja; Mukherjee, Ali; Leitzel, Kim; Kostler, Wolfgang J; Lipton, Allan; Bates, Michael; Arteaga, Carlos L

    2011-03-01

    In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab. PMID:21324925

  3. Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion*

    PubMed Central

    Flanagan, John J.; Mukherjee, Indrani; Barlowe, Charles

    2015-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes. PMID:25750128

  4. Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers

    SciTech Connect

    Sommerhalter, M.; Voegtli, W.C.; Perlstein, D.L.; Ge, J.; Stubbe, J.; Rosenzweig, A.C.

    2010-03-08

    Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix {alpha}B, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices {alpha}A and {alpha}3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.

  5. 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.

  6. Single low-dose cyclophosphamide combined with interleukin-12 gene therapy is superior to a metronomic schedule in inducing immunity against colorectal carcinoma in mice

    PubMed Central

    Malvicini, Mariana; Alaniz, Laura; Bayo, Juan; Garcia, Mariana; Piccioni, Flavia; Fiore, Esteban; Atorrasagasti, Catalina; Aquino, Jorge B.; Matar, Pablo; Mazzolini, Guillermo

    2012-01-01

    The use of conventional cytotoxic agents at metronomic schedules, alone or in combination with targeted agents or immunotherapy, is being explored as a promising anticancer strategy. We previously reported a potent antitumor effect of a single low-dose cyclophosphamide and interleukin-12 (IL-12) gene therapy against advanced gastrointestinal carcinoma, in mice. Here, we assessed whether the delivery of IL-12 by gene therapy together with metronomic cyclophosphamide exerts antitumor effects in a murine model of colorectal carcinoma. This combination therapy was able, at least in part, to reverse immunosuppression, by decreasing the number of regulatory T cells (Tregs) as well as of splenic myeloid-derived suppressor cells (MDSCs). However, metronomic cyclophosphamide plus IL-12 gene therapy failed to increase the number of tumor-infiltrating T lymphocytes and, more importantly, to induce a specific antitumor immune response. With respect to this, cyclophosphamide at a single low dose displayed a superior anticancer profile than the same drug given at a metronomic schedule. Our results may have important implications in the design of new therapeutic strategies against colorectal carcinoma using cyclophosphamide in combination with immunotherapy. PMID:23170252

  7. Preclinical Evaluation of Oncolytic Δγ134.5 Herpes Simplex Virus Expressing Interleukin-12 for Therapy of Breast Cancer Brain Metastases

    PubMed Central

    Cody, James J.; Scaturro, Pietro; Cantor, Alan B.; Yancey Gillespie, G.; Parker, Jacqueline N.; Markert, James M.

    2012-01-01

    The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ134.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including “triple-negative” and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ134.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases. PMID:23346408

  8. Encapsulation and Characterization of Proton-Bound Amine Homodimers in a Water Soluble, Self-Assembled Supramolecular Host

    SciTech Connect

    Pluth, Michael; Fiedler, Dorothea; Mugridge, Jeffrey; Bergman, Robert; Raymond, Kenneth

    2008-10-01

    Cyclic amines can be encapsulated in a water-soluble self-assembled supramolecular host upon protonation. The hydrogen bonding ability of the cyclic amines, as well as the reduced degrees of rotational freedom, allows for the formation of proton-bound homodimers inside of the assembly which are otherwise not observable in aqueous solution. The generality of homodimer formation was explored with small N-alkyl aziridines, azetidines, pyrrolidines and piperidines. Proton-bound homodimer formation is observed for N-alkylaziridines (R = methyl, isopropyl, tert-butyl), N-alkylazetidines (R = isopropyl, tertbutyl), and N-methylpyrrolidine. At high concentration, formation of a proton-bound homotrimer is observed in the case of N-methylaziridine. The homodimers stay intact inside the assembly over a large concentration range, thereby suggesting cooperative encapsulation. Both G3(MP2)B3 and G3B3 calculations of the proton-bound homodimers were used to investigate the enthalpy of the hydrogen bond in the proton-bound homodimers and suggest that the enthalpic gain upon formation of the proton-bound homodimers may drive guest encapsulation.

  9. From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

    PubMed Central

    Gresock, Michael G.; Kastead, Kyle A.

    2015-01-01

    ABSTRACT The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them. IMPORTANCE Canonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport

  10. Depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma

    PubMed Central

    Thaci, B; Ahmed, AU; Ulasov, IV; Wainwright, DA; Nigam, P; Auffinger, B; Tobias, AL; Han, Y; Zhang, L; Moon, K-S; Lesniak, MS

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) accumulate in the glioma microenvironment during tumor progression and promote immunosuppression. Interleukin-12 (IL-12) immunogene therapy can alter MDSCs toward an antigen-presenting cell phenotype and these mature cells can have a central role in antigen presentation. It remains unclear, however, how MDSC depletion can affect glioma immunotherapy. In this study, we generated a replication-deficient adenoviral vector, Ad.5/3.cRGD-mIL12p70, that transduces the GL261-based murine glioma cell line, resulting in the induction of biologically active, murine IL12p70 expression. Ex vivo, IL-12 expressed by GL261 cells induced interferon-γ synthesis in CD8 +T cells (P<0.001), CD4 +T cells (P =0.009) and natural killer cells (P =0.036). When injected 1 week after tumor implantation, Ad.5/3.cRGD-mIL12p70 successfully prolonged the survival of glioma-bearing mice. Sixty percent of animals treated with IL-12 immunotherapy were long-term survivors over 175 days, whereas all the control group animals expired by 40 days after tumor implantation (P =0.026). Mice receiving Ad.5/3.cRGD-mIL12p70 also accumulated 50% less MDSCs in the brain than the control group (P =0.007). Moreover, in the IL-12 group, MDSCs significantly overexpressed CD80 and major histocompatibility complex class II molecules (P =0.041). Depletion of MDSCs with Gr1 +antibody had no survival benefit induced by IL-12-mediated immunotherapy. Of note, IL-12 therapy increased the presence of myeloid dendritic cells (mDCs) in the glioma microenvironment (P =0.0069). Ultimately, the data show that in the context of IL-12 immunogene therapy, MDSCs are dispensable and mDCs may provide the majority of antigen presentation in the brain. PMID:24434573

  11. Depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma.

    PubMed

    Thaci, B; Ahmed, A U; Ulasov, I V; Wainwright, D A; Nigam, P; Auffinger, B; Tobias, A L; Han, Y; Zhang, L; Moon, K-S; Lesniak, M S

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) accumulate in the glioma microenvironment during tumor progression and promote immunosuppression. Interleukin-12 (IL-12) immunogene therapy can alter MDSCs toward an antigen-presenting cell phenotype and these mature cells can have a central role in antigen presentation. It remains unclear, however, how MDSC depletion can affect glioma immunotherapy. In this study, we generated a replication-deficient adenoviral vector, Ad.5/3.cRGD-mIL12p70, that transduces the GL261-based murine glioma cell line, resulting in the induction of biologically active, murine IL12p70 expression. Ex vivo, IL-12 expressed by GL261 cells induced interferon-γ synthesis in CD8(+) T cells (P<0.001), CD4(+) T cells (P=0.009) and natural killer cells (P=0.036). When injected 1 week after tumor implantation, Ad.5/3.cRGD-mIL12p70 successfully prolonged the survival of glioma-bearing mice. Sixty percent of animals treated with IL-12 immunotherapy were long-term survivors over 175 days, whereas all the control group animals expired by 40 days after tumor implantation (P=0.026). Mice receiving Ad.5/3.cRGD-mIL12p70 also accumulated 50% less MDSCs in the brain than the control group (P=0.007). Moreover, in the IL-12 group, MDSCs significantly overexpressed CD80 and major histocompatibility complex class II molecules (P=0.041). Depletion of MDSCs with Gr1(+) antibody had no survival benefit induced by IL-12-mediated immunotherapy. Of note, IL-12 therapy increased the presence of myeloid dendritic cells (mDCs) in the glioma microenvironment (P=0.0069). Ultimately, the data show that in the context of IL-12 immunogene therapy, MDSCs are dispensable and mDCs may provide the majority of antigen presentation in the brain. PMID:24434573

  12. 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

  13. Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

    PubMed

    Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

    2015-01-01

    The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. PMID:25583174

  14. Silibinin Inhibits Ultraviolet B Radiation-Induced DNA-Damage and Apoptosis by Enhancing Interleukin-12 Expression in JB6 Cells and SKH-1 Hairless Mouse Skin

    PubMed Central

    Narayanapillai, Sreekanth; Agarwal, Chapla; Deep, Gagan; Agarwal, Rajesh

    2013-01-01

    Recent studies have demonstrated silibinin efficacy against ultraviolet B (UVB)-induced skin carcinogenesis via different mechanisms in cell lines and animal models; however, its role in regulating interleukin-12 (IL-12), an immunomodulatory cytokine that reduces UVB-induced DNA damage and apoptosis, is not known. Here, we report that UVB irradiation causes caspase 3 and PARP cleavage and apoptosis, and addition of recombinant IL-12 or silibinin immediately after UVB significantly protects UVB-induced apoptosis in JB6 cells. IL-12 antibody-mediated blocking of IL-12 activity compromised the protective effects of both IL-12 and silibinin. Both silibinin and IL-12 also accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in mouse skin epidermis, and that similar to cell-culture findings, silibinin topical application immediately after UVB exposure causes a strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs. Together, these findings for the first time provide an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. In view of the fact that an enhanced endogenous IL-12 level could effectively remove UVB-caused DNA damage and associated skin cancer, our findings suggest that the use of silibinin in UVB-damaged human skin would also be a practical and translational strategy to manage solar radiation-caused skin damages as well as skin cancer. PMID:23359305

  15. Chronic Helminth Infection Induces Alternatively Activated Macrophages Expressing High Levels of CCR5 with Low Interleukin-12 Production and Th2-Biasing Ability

    PubMed Central

    Rodríguez-Sosa, Miriam; Satoskar, Abhay R.; Calderón, Rodrigo; Gomez-Garcia, Lorena; Saavedra, Rafael; Bojalil, Rafael; Terrazas, Luis I.

    2002-01-01

    Helminth infections induce Th2-type biased immune responses. Although the mechanisms involved in this phenomenon are not yet clearly defined, antigen-presenting cells (APC) could play an important role in this process. Here, we have used peritoneal macrophages (F4/80+) recruited at different times after challenge with Taenia crassiceps as APC and tested their ability to regulate Th1/Th2 differentiation. Macrophages from acute infections produced high levels of interleukin-12 (IL-12) and nitric oxide (NO), paralleled with low levels of IL-6 and prostaglandin E2 (PGE2) and with the ability to induce strong antigen-specific CD4+ T-cell proliferation in response to nonrelated antigens. In contrast, macrophages from chronic infections produced higher levels of IL-6 and PGE2 and had suppressed production of IL-12 and NO, associated with a poor ability to induce antigen-specific proliferation in CD4+ T cells. Failure to induce proliferation was not due to a deficient expression of accessory molecules, since major histocompatibility complex class II, CD40, and B7-2 were up-regulated, together with CD23 and CCR5 as infection progressed. These macrophages from chronic infections were able to bias CD4+ T cells to produce IL-4 but not gamma interferon (IFN-γ), contrary to macrophages from acute infections. Blockade of B7-2 and IL-6 and inhibition of PGE2 failed to restore the proliferative response in CD4+ T cells. Furthermore, studies using STAT6−/− mice revealed that STAT6-mediated signaling was essential for the expansion of these alternatively activated macrophages. These data demonstrate that helminth infections can induce different macrophage populations that have Th2-biasing properties. PMID:12065507

  16. Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity.

    PubMed Central

    Vizler, C.; Rosato, A.; Calderazzo, F.; Quintieri, L.; Fruscella, P.; Wainstok de Calmanovici, R.; Mantovani, A.; Vecchi, A.; Zanovello, P.; Collavo, D.

    1998-01-01

    In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis. PMID:9484826

  17. Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

    PubMed

    Lasek, W; Golab, J; Maśliński, W; Switaj, T; Bałkowiec, E Z; Stokłosa, T; Giermasz, A; Malejczyk, M; Jakóbisiak, M

    1999-09-01

    Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction. PMID:10477391

  18. Altered interleukin-12 responsiveness in Th1 and Th2 cells is associated with the differential activation of STAT5 and STAT1.

    PubMed

    Gollob, J A; Murphy, E A; Mahajan, S; Schnipper, C P; Ritz, J; Frank, D A

    1998-02-15

    T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1alpha, and STAT1beta, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12-induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12-induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-gamma (IFN-gamma) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-gamma compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12-induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-gamma is not a direct effect of IFN-gamma on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-gamma. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-gamma production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells. PMID:9454765

  19. Interleukin-12-Producing CD103+ CD11b− CD8+ Dendritic Cells Are Responsible for Eliciting Gut Intraepithelial Lymphocyte Response against Encephalitozoon cuniculi

    PubMed Central

    Moretto, Magali M.; Harrow, Danielle I.; Hawley, Teresa S.

    2015-01-01

    Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b− CD8+ DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b− CD8+ DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103+ CD11b− CD8+ DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103+ CD11b− CD8+ DC isolated from infected animals in the generation of an IEL response against a live pathogen. PMID:26416905

  20. Polydnavirus Ank Proteins Bind NF-κB Homodimers and Inhibit Processing of Relish

    PubMed Central

    Strand, Michael R.

    2012-01-01

    Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-κB (NF-κB) transcription factors, and inhibitor κB (IκB) proteins that complex with and regulate NF-κBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IκBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IκB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IκBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IκB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and –N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IκB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and –N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-κB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IκB binding affinities, while also showing that viral mimics disable Relish processing. PMID:22654665

  1. A Role for the PERIOD:PERIOD Homodimer in the Drosophila Circadian Clock

    PubMed Central

    Wolf, Eva; Stanewsky, Ralf

    2009-01-01

    Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of ∼24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function. PMID:19402744

  2. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore

    PubMed Central

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H.; Hustedt, Eric J.; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  3. Synthesis of diketopiperazine-based carboline homodimers and in vitro growth inhibition of human carcinomas.

    PubMed

    Deveau, Amy M; Costa, Nancy E; Joshi, Elizabeth M; Macdonald, Timothy L

    2008-06-15

    Starting from d- or l-tryptophan, we have synthesized and characterized six compounds 2.29-2.31a and b that belong to a class of nitrogen heterocycles: the carboline-based homodimers. Each individual homodimer features a 1,3-trans relationship on each side of the central diketopiperazine core, but differs in absolute stereochemistry and also in substitution on the 4' and 4'' oxygens (-Bn, -CH(3), or -H). The in vitro cytotoxicity of the six compounds was evaluated by measuring the growth inhibition in NCI-H520 and PC-3 human carcinoma cells. Phenol 2.30a inhibited cancer cell growth approximately three times better than its enantiomer 2.30b and possessed a GI(50) comparable to the clinically used agent etoposide in both cell lines. We have concluded that both the stereochemistry imparted by l-tryptophan and the presence of hydroxy substituents at the 4' and 4'' positions are necessary to generate cytotoxic properties in the homodimer class. We are now employing 2.30a as a new lead compound in our efforts to discover improved indole-based cancer chemotherapeutics. PMID:18502124

  4. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.

    PubMed

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H; Hustedt, Eric J; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the 'α3/α5' interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known 'α6:α6' interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  5. HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

    PubMed Central

    Basile, Lena A.; Ellefson, Dolph; Gluzman-Poltorak, Zoya; Junes-Gill, Katiana; Mar, Vernon; Mendonca, Sarita; Miller, Joseph D.; Tom, Jamie; Trinh, Alice; Gallaher, Timothy K.

    2012-01-01

    HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting

  6. Differences in binding behavior of (-)-epigallocatechin gallate to β-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B).

    PubMed

    Keppler, Julia K; Martin, Dierk; Garamus, Vasil M; Schwarz, Karin

    2015-11-01

    The lipocalin β-lactoglobulin (β-LG) exists in different natural genetic variants--of which β-LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes--building homodimers of β-LG A and β-LG B and heterodimers of β-LG AB. Although β-LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero- and homodimers of β-LG is largely unknown. The present findings revealed significant differences for hetero- and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (-)-epigallocatechin gallate (EGCG)). These findings were confirmed using FT-IR, where the addition of EGCG influenced the β-sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of β-LG in the order β-LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of β-LG and gives not only important information for β-LG binding studies, but may also apply for polymorphisms of other self-aggregating lipocalins. PMID:26038095

  7. PMLRAR homodimers: distinct DNA binding properties and heteromeric interactions with RXR.

    PubMed Central

    Perez, A; Kastner, P; Sethi, S; Lutz, Y; Reibel, C; Chambon, P

    1993-01-01

    Fusion proteins (named PMLRAR) between PML and the retinoic acid receptor alpha (RAR alpha) are generated as a result of the t(15;17) chromosomal translocation found in acute promyelocytic leukemia (APL). We show here that PMLRAR proteins exist in solution as stable homodimers whose formation is mediated by a presumptive coiled coil in the PML moiety. In contrast to RAR alpha, which requires heterodimerization with RXR for efficient DNA binding, PMLRAR homodimers can bind to target sequences in the absence of RXR, and the binding pattern of PMLRAR homodimeric complexes to directly repeated motif (DR) response elements with 1-5 bp spacers is different from that of RAR/RXR heterodimeric complexes. We show that the presence of RXR induces the formation of PMLRAR/RXR heteromeric complexes which bind to DNA via one RAR DNA binding domain (DBD) and one RXR DBD, like 'classical' RAR/RXR heterodimers. PMLRAR interaction with RXR occurs in solution and in transfected cultured Cos cells, and PMLRAR is able to sequester RXR efficiently in the cytoplasm, suggesting that dominant 'inactivation' of RXR may be a possible mechanism of action for PMLRAR. Accordingly, we show that PMLRAR can both prevent the binding of the vitamin D3 receptor (VDR) to a target sequence in vitro and inhibit vitamin D3-dependent activation of a VDR-responsive reporter gene in transfected cells. These results suggest that both the distinct DNA binding properties of PMLRAR homodimers and the sequestration of RXR by PMLRARs may contribute to the molecular mechanisms which underlie the pathogenesis of APL. We also report that RXR alpha transcripts are down-regulated by RA-treatment in promyelocytic cells. Images PMID:8393784

  8. Structure-based network analysis of an evolved G protein-coupled receptor homodimer interface

    PubMed Central

    Nichols, Sara E; Hernández, Carlos X; Wang, Yi; McCammon, James Andrew

    2013-01-01

    Crystallographic structures and experimental assays of human CXC chemokine receptor type 4 (CXCR4) provide strong evidence for the capacity to homodimerize, potentially as a means of allosteric regulation. Even so, how this homodimer forms and its biological significance has yet to be fully characterized. By applying principles from network analysis, sequence-based approaches such as statistical coupling analysis to determine coevolutionary residues, can be used in conjunction with molecular dynamics simulations to identify residues relevant to dimerization. Here, the predominant coevolution sector lies along the observed dimer interface, suggesting functional relevance. Furthermore, coevolution scoring provides a basis for determining significant nodes, termed hubs, in the network formed by residues found along the interface of the homodimer. These node residues coincide with hotspots indicating potential druggability. Drug design efforts targeting such key residues could potentially result in modulation of binding and therapeutic benefits for disease states, such as lung cancers, lymphomas and latent HIV-1 infection. Furthermore, this method may be applied to any protein–protein interaction. PMID:23553730

  9. Synthesis and activity of novel homodimers of Morita-Baylis-Hillman adducts against Leishmania donovani: A twin drug approach.

    PubMed

    da Silva, Wagner A V; Rodrigues, Daniele C; de Oliveira, Ramon G; Mendes, Rhuan K S; Olegário, Tayná R; Rocha, Juliana C; Keesen, Tatjana S L; Lima-Junior, Claudio G; Vasconcellos, Mário L A A

    2016-09-15

    It is reported here the synthesis of novel Homodimers 12-19 of Morita-Baylis-Hillman adducts (MBHA) from one-pot Morita-Baylis-Hillman Reaction (MBHR) between aromatic aldehydes as eletrophiles and ethylene glycol diacrylate as Michael acceptor (35-94% yields) using cheap and green conditions. The bioactivities were evaluated against promastigote form of Leishmania donovani. All homodimers showed to be more potent than corresponding monomers. It is worth highlighting that the halogenated homodimers 17 and 18 (0.50μM) is almost 400 times more active than the corresponding monomer 10 and 1.24 times more potent than the second-line drug amphotericin B (0.62μM). Moreover, the selectivity index to 18 is very high (SIrb>400) far better than amphotericin B (SIrb=18.73). This is the first report of twin drugs strategy applied on Morita-Baylis-Hillman adducts. PMID:27520941

  10. A Novel Styryldehydropyridocolinium Homodimer: Synthesis and Fluorescence Properties Upon Interaction with DNA.

    PubMed

    Yao, Huirong; Chang, Lifang; Liu, Chang; Jiao, Xiaojie; He, Song; Liu, Haijun; Zeng, Xianshun

    2015-11-01

    A novel homodimer of the styryldehydropyridocolinium dye (TPTP) has been synthesized and characterized. Free TPTP exhibited low fluorescence quantum yield and large Stokes shift (over 160 nm) in water. However, it showed a significant fluorescence turn-on effect upon intercalation into DNA base pairs. Meanwhile, the fluorescence intensity of the intercalated structures formed by TPTP and DNA decreased quickly upon addition of deoxyribonuclease I, indicating that the dye can be used to monitor deoxyribonuclease I activity and DNA hydrolysis. Electrophoresis analysis revealed that the dye had intercalative binding to DNA and can potentially be used for DNA staining in electrophoresis. Thus, the innate nature of large Stokes shift and excellent fluorescence turn on effect upon interaction with DNA endue the dye with a wide range of applications. PMID:26384336

  11. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis

    SciTech Connect

    Hsueh, A.J.W.; Dahl, K.D.; Vaughan, J.; Tucker, E.; Rivier, J.; Bardin, C.W.; Vale, W.

    1987-07-01

    Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an ..cap alpha beta.. heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the ..cap alpha beta.. heterodimer, the ..beta beta.. homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrated the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, the authors have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the ..cap alpha beta.. heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the ..beta beta.. homodimer suppresses androgen production. The data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops.

  12. Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution.

    PubMed

    Bilgrami, Sameeta; Tomar, Shailly; Yadav, Savita; Kaur, Punit; Kumar, Janesh; Jabeen, Talat; Sharma, Sujata; Singh, Tej P

    2004-08-13

    This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway. PMID:15317139

  13. The United Stirling P40 engine for solar dish concentrator application

    NASA Technical Reports Server (NTRS)

    Ortegren, L.; Sjostedt, L. E.

    1980-01-01

    The United Stirling P40 engine is a key component in a solar concentration based energy conversion system, to be demonstrated and tested during 1980-81. The inherent characteristics of modern Stirling engines is reviewed focusing on the baseline P40 double-acting engine. The extent of modifications required for the solar application is reviewed and performance data are predicted. Finally, the potential of an advanced solar Stirling engine is briefly considered.

  14. Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium

    PubMed Central

    Cheung, Lydia WT; Walkiewicz, Katarzyna W; Besong, Tabot MD; Guo, Huifang; Hawke, David H; Arold, Stefan T; Mills, Gordon B

    2015-01-01

    The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer–dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development. DOI: http://dx.doi.org/10.7554/eLife.06866.001 PMID:26222500

  15. Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

    SciTech Connect

    Der, Bryan S.; Machius, Mischa; Miley, Michael J.; Mills, Jeffrey L.; Szyperski, Thomas; Kuhlman, Brian

    2015-10-15

    Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 {micro}M. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (C{alpha} rmsd = 1.4 {angstrom}).

  16. Secreted Progranulin Is a Homodimer and Is Not a Component of High Density Lipoproteins (HDL)*

    PubMed Central

    Nguyen, Andrew D.; Nguyen, Thi A.; Cenik, Basar; Yu, Gang; Herz, Joachim; Walther, Tobias C.; Davidson, W. Sean; Farese, Robert V.

    2013-01-01

    Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170–180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180–190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL. PMID:23364791

  17. Plasmon-mediated binding forces on gold or silver homodimer and heterodimer

    NASA Astrophysics Data System (ADS)

    Liaw, Jiunn-Woei; Kuo, Ting-Yu; Kuo, Mao-Kuen

    2016-02-01

    This study theoretically investigates plasmon-mediated optical binding forces, which are exerted on metal homo or heterodimers, induced by the normal illumination of a linearly polarized plane wave or Gaussian beam. Using the multiple multipole method, we analyzed the optical force in terms of Maxwell's stress tensor for various interparticle distance at some specific wavelengths. Numerical results show that for a given wavelength there are several stable equilibrium distances between two nanoparticles (NPs) of a homodimer, which are slightly shorter than some integer multiples of the wavelength in medium, such that metal dimer acts as bonded together. At these specific interparticle distances, the optical force between dimer is null and serves a restoring force, which is repulsive and attractive, respectively, as the two NPs are moving closer to and away from each other. The spring constant of the restoring force at the first stable equilibrium is always the largest, indicating that the first stable equilibrium distance is the most stable one. Moreover, the central line (orientation) of a dimer tends to be perpendicular to the polarization of light. For the cases of heterodimers, the phenomenon of stable equilibrium interparticle distance still exists, except there is an extra net photophoretic force drifting the heterodimer as one. Moreover, gradient force provided by a Gaussian beam may reduce the stability of these equilibriums, so larger NPs are preferred to stabilize a dimer under illumination of Gaussian beam. The finding may pave the way for using optical manipulation on the gold or silver colloidal self-assembly.

  18. Photoelectron spectroscopic and density functional theoretical studies of the 2'-deoxycytidine homodimer radical anion.

    PubMed

    Storoniak, Piotr; Rak, Janusz; Ko, Yeon Jae; Wang, Haopeng; Bowen, Kit H

    2013-08-21

    The intact (parent) 2'-deoxycytidine homodimer anion, (dC)2 (●-), was generated in the gas phase (in vacuo) using an infrared desorption∕photoemission source and its photoelectron spectrum was recorded using a pulsed, magnetic bottle photoelectron spectrometer. The photoelectron spectrum (PES) revealed a broad peak with the maximum at an electron binding energy between 1.6 and 1.9 eV and with a threshold at ∼1.2 eV. The relative energies and vertical detachment energies of possible anion radicals were calculated at the B3LYP/6-31++G(∗∗) level of theory. The most stable anion radicals are the complexes involving combinations of the sugar[middle dot][middle dot][middle dot]base and base[middle dot][middle dot][middle dot]base interactions. The calculated adiabatic electron affinities and vertical detachment energies of the most stable (dC)2 (●-) anions agree with the experimental values. In contrast with previous experimental-computational studies on the anionic complexes involving nucleobases with various proton-donors, the electron-induced proton transferred structures of (dC)2 (●-) are not responsible for the shape of PES. PMID:23968113

  19. Photoelectron spectroscopic and density functional theoretical studies of the 2'-deoxycytidine homodimer radical anion

    NASA Astrophysics Data System (ADS)

    Storoniak, Piotr; Rak, Janusz; Ko, Yeon Jae; Wang, Haopeng; Bowen, Kit H.

    2013-08-01

    The intact (parent) 2'-deoxycytidine homodimer anion, (dC)2•-, was generated in the gas phase (in vacuo) using an infrared desorption/photoemission source and its photoelectron spectrum was recorded using a pulsed, magnetic bottle photoelectron spectrometer. The photoelectron spectrum (PES) revealed a broad peak with the maximum at an electron binding energy between 1.6 and 1.9 eV and with a threshold at ˜1.2 eV. The relative energies and vertical detachment energies of possible anion radicals were calculated at the B3LYP/6-31++G** level of theory. The most stable anion radicals are the complexes involving combinations of the sugar...base and base...base interactions. The calculated adiabatic electron affinities and vertical detachment energies of the most stable (dC)2•- anions agree with the experimental values. In contrast with previous experimental-computational studies on the anionic complexes involving nucleobases with various proton-donors, the electron-induced proton transferred structures of (dC)2•- are not responsible for the shape of PES.

  20. The infrared band intensities and other properties of the homodimers of the methyl and silyl halides: An ab initio study

    NASA Astrophysics Data System (ADS)

    Ford, Thomas A.

    2012-02-01

    The properties of the homodimers of methyl and silyl fluoride, chloride and bromide have been determined by means of ab initio molecular orbital calculations. The interaction energies, molecular structures, vibrational spectra and molecular orbital properties have been investigated, and some common features within each family have been observed. A number of systematic differences in the properties of the dimers have also been noted and rationalized. Typically, discussion of the results of such calculations has focused on the vibrational wavenumber shifts occurring on complexation, and the accompanying changes in the infrared band intensities have received relatively little attention. This paper aims to reposition infrared intensities as valid and useful parameters with which to interpret the formation of the homodimers of polar molecules.

  1. Native Serotonin 5-HT2C Receptors Are Expressed as Homodimers on the Apical Surface of Choroid Plexus Epithelial Cells

    PubMed Central

    Grinde, Ellinor; Lindsley, Tara; Teitler, Milt; Mancia, Filippo; Cowan, Ann; Mazurkiewicz, Joseph E.

    2015-01-01

    G protein–coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti–5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10−9 cm2/s and were expressed at 32 receptors/μm2 on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit. PMID:25609374

  2. Native serotonin 5-HT2C receptors are expressed as homodimers on the apical surface of choroid plexus epithelial cells.

    PubMed

    Herrick-Davis, Katharine; Grinde, Ellinor; Lindsley, Tara; Teitler, Milt; Mancia, Filippo; Cowan, Ann; Mazurkiewicz, Joseph E

    2015-04-01

    G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/μm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit. PMID:25609374

  3. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis.

    PubMed Central

    Hsueh, A J; Dahl, K D; Vaughan, J; Tucker, E; Rivier, J; Bardin, C W; Vale, W

    1987-01-01

    Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an alpha beta heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the alpha beta heterodimer, the beta beta homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrating the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, we have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the alpha beta heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the beta beta homodimer suppresses androgen production. Furthermore, similar modulatory actions of inhibin-related proteins were found in cultured ovarian theca-interstitial cells and theca explants treated with LH. In contrast, treatment with the inhibin-related proteins alone did not affect gonadal steroidogenesis. Our data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops. PMID:3474640

  4. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  5. Further Studies on the Origins of Asymmetric Charge Partitioning in Protein Homodimers

    PubMed Central

    Jurchen, John C.; Garcia, David E.; Williams, Evan R.

    2005-01-01

    Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of α-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some

  6. Comparison of Cyclooxygenase-1 Crystal Structures: Cross-Talk between Monomers Comprising Cyclooxygenase-1 Homodimers

    SciTech Connect

    Sidhu, Ranjinder S.; Lee, Jullia Y.; Yuan, Chong; Smith, William L.

    2010-11-01

    Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 (also called cyclooxygenases (COXs)-1 and -2) catalyze the committed step in prostaglandin biosynthesis. Both isoforms are targets of nonsteroidal antiinflammatory drugs (NSAIDs). PGHSs are homodimers that exhibit half-of-sites COX activity; moreover, some NSAIDs cause enzyme inhibition by binding only one monomer. To learn more about the cross-talk that must be occurring between the monomers comprising each PGHS-1 dimer, we analyzed structures of PGHS-1 crystallized under five different conditions including in the absence of any tightly binding ligand and in the presence of nonspecific NSAIDs and of a COX-2 inhibitor. When crystallized with substoichiometric amounts of an NSAID, both monomers are often fully occupied with inhibitor; thus, the enzyme prefers to crystallize in a fully occupied form. In comparing the five structures, we only observe changes in the positions of residues 123-129 and residues 510-515. In cases where one monomer is fully occupied with an NSAID and the partner monomer is incompletely occupied, an alternate conformation of the loop involving residues 123-129 is seen in the partially occupied monomer. We propose, on the basis of this observation and previous cross-linking studies, that cross-talk between monomers involves this mobile 123-129 loop, which is located at the dimer interface. In ovine PGHS-1 crystallized in the absence of an NSAID, there is an alternative route for substrate entry into the COX site different than the well-known route through the membrane binding domain.

  7. IFN-γ induction by neutrophil-derived IL-17A homodimer augments pulmonary antibacterial defense.

    PubMed

    Cai, S; Batra, S; Langohr, I; Iwakura, Y; Jeyaseelan, S

    2016-05-01

    The role of interleukin-17A (IL-17A) in host defense against Legionella pneumophila remains elusive. To address this issue, we used Il17a(-/-), Il17f(-/-), and Il17a/Il17f(-/-) mice on a C57Bl/6 (non-permissive) background and IL-17 neutralizing Abs in mice on an A/J (permissive) background. Higher bacterial (L. pneumophila) counts in the lung and blood along with reduced neutrophil recruitment were detected in Il17a(-/-), but not Il17f(-/-), mice. We found that neutrophils produce IL-17A homodimer (IL-17A) during L. pneumophila infection, and hematopoietic cell-derived IL-17A is known to be important for bacterial clearance. Thus, intratracheal administration of wild-type neutrophils or recombinant IL-17A restored bacterial clearance and neutrophil recruitment in Il17a(-/-) mice. Furthermore, neutrophil-depleted Rag2(-/-) and Rag2/Il-2rγ(-/-) mice exhibited increased bacterial burden, reduced neutrophil influx and IL-17A production in the lung. Recombinant IFN-γ administration in Il17a(-/-) mice augmented bacterial elimination, whereas IL-17A administration in Ifnγ(-/-) mice did not augment bacterial clearance. IFN-γ is produced by T cells, but not neutrophils or macrophages, suggesting that neutrophil-derived IL-17A induces IFN-γ in a paracrine fashion. Human pneumonic lungs and human neutrophils challenged with L. pneumophila exhibited increased numbers of IL-17A producing cells. These findings display a novel function of neutrophil-derived IL-17A in antibacterial defense via the induction of IFN-γ in a paracrine manner. PMID:26349661

  8. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.

    PubMed

    Dong, Liang; Sharma, Narayan P; Jurban, Brice J; Smith, William L

    2013-10-01

    Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing. PMID:23955344

  9. Human cyclooxygenase-2 is a sequence homodimer that functions as a conformational heterodimer.

    PubMed

    Dong, Liang; Vecchio, Alex J; Sharma, Narayan P; Jurban, Brice J; Malkowski, Michael G; Smith, William L

    2011-05-27

    Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H(2). Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E(cat)) and an allosteric monomer (E(allo)). Heme binds tightly only to the peroxidase site of E(cat), whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E(cat). E(cat) is regulated by E(allo) in a manner dependent on what ligand is bound to E(allo). Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E(allo). AA can bind to E(cat) and E(allo), but the affinity of AA for E(allo) is 25 times that for E(cat). Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E(allo) in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E(cat) or E(allo). Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both base-line prostanoid synthesis and responses to COX inhibitors. PMID:21467029

  10. Comparison of Cyclooxygenase-1 Crystal Structures: Cross-Talk Between Monomers Comprising Cyclooxygenase-1 Homodimers

    PubMed Central

    Sidhu, Ranjinder S.; Lee, Jullia Y.; Yuan, Chong; Smith, William L.

    2010-01-01

    Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 (also called cyclooxygenases (COXs)-1 and -2) catalyze the committed step in prostaglandin biosynthesis. Both isoforms are targets of nonsteroidal anti-inflammatory drugs (NSAIDs). PGHSs are homodimers that exhibit half-of-sites COX activity; moreover, some NSAIDs cause enzyme inhibition by binding only one monomer. To learn more about the cross-talk that must be occurring between the monomers comprising each PGHS-1 dimer, we analyzed structures of PGHS-1 crystallized under five different conditions including in the absence of any tightly binding ligand and in the presence of non-specific NSAIDs and of a COX-2 inhibitor. When crystallized with sub-stoichiometric amounts of an NSAID, both monomers are often fully occupied with inhibitor; thus, the enzyme prefers to crystallize in a fully occupied form. In comparing the five structures, we only observe changes in the positions of residues 123-129 and residues 510-515. In cases where one monomer is fully occupied with an NSAID and the partner monomer is incompletely occupied, an alternate conformation of the loop involving residues 123-129 is seen in the partially occupied monomer. We propose, based on this observation and previous cross-linking studies, that cross-talk between monomers involves this mobile 123-129 loop, which is located at the dimer interface. In ovine PGHS-1 crystallized in the absence of an NSAID, there is an alternative route for substrate entry into the COX site different than the well-known route through the membrane binding domain. PMID:20669977

  11. Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer*

    PubMed Central

    Dong, Liang; Sharma, Narayan P.; Jurban, Brice J.; Smith, William L.

    2013-01-01

    Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing. PMID:23955344

  12. Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

    SciTech Connect

    Lashkov, A. A.; Zhukhlistova, N. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2009-03-15

    The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium (St UPh) was determined with high accuracy. The structure was refined at 1.80 A resolution to R{sub work} = 16.1% and R{sub free} = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 A, 1.042{sup o}, and 0.071{sup o}, respectively. The coordinate error estimated by the Luzzati plot is 0.166 A. The coordinate error based on the maximum likelihood is 0.199 A. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K{sup +} ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

  13. Role of Anthocyanin-enriched Purple-fleshed Sweet Potato P40 in Colorectal Cancer Prevention

    PubMed Central

    Lim, Soyoung; Xu, Jianteng; Kim, Jaeyong; Chen, Tzu-Yu; Su, Xiaoyu; Standard, Joseph; Carey, Edward; Griffin, Jason; Herndon, Betty; Katz, Benjamin; Tomich, John; Wang, Weiqun

    2013-01-01

    Scope Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention. However, the content of anthocyanins in staple foods is typically low and the mechanisms by which they exert anti-cancer activity is not yet fully defined. Methods and results We selected an anthocyanin-enriched purple-fleshed sweet potato clone, P40, and investigated its potential anti-cancer effect in both in vitro cell culture and in vivo animal model. In addition to a high level of total phenolics and antioxidant capacity, P40 possesses a high content of anthocyanins at 7.5 mg/g dry matter. Treatment of human colonic SW480 cancer cells with P40 anthocyanin extracts at 0–40 μM of peonidin-3-glucoside equivalent resulted in a dose-dependent decrease in cell number due to cytostatic arrest of cell cycle at G1 phase but not cytotoxicity. Furthermore, dietary P40 at 10–30% significantly suppressed azoxymethane-induced formation of aberrant crypt foci in the colons of CF-1 mice in conjunction with, at least in part, a lesser proliferative PCNA and a greater apoptotic caspase-3 expression in the colon mucosal epithelial cells. Conclusion These observations, coupled with both in vitro and in vivo studies reported here, suggest anthocyanin-enriched sweet potato P40 may protect against colorectal cancer by inducing cell cycle arrest, anti-proliferative and apoptotic mechanisms. PMID:23784800

  14. Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23

    PubMed Central

    Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D. Brent; Monedero, Vicente

    2011-01-01

    The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria PMID:21178363

  15. Functional analysis of the p40 and p75 proteins from Lactobacillus casei BL23.

    PubMed

    Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D Brent; Monedero, Vicente

    2010-01-01

    The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria. PMID:21178363

  16. Crystallization and preliminary crystallographic analysis of p40{sup phox}, a regulatory subunit of NADPH oxidase

    SciTech Connect

    Honbou, Kazuya; Yuzawa, Satoru; Suzuki, Nobuo N.; Fujioka, Yuko; Sumimoto, Hideki; Inagaki, Fuyuhiko

    2006-10-01

    Human p40{sup phox} was expressed, purified and crystallized. Diffraction data were collected to a resolution of 3.0 Å. p40{sup phox} is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for production of the superoxide that kills invasive microorganisms. Full-length p40{sup phox} was expressed in Escherichia coli, purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 3.0 Å resolution at 100 K using synchrotron radiation. The crystal belongs to space group C222{sub 1}, with unit-cell parameters a = 146.27, b = 189.81, c = 79.88 Å. This crystal was estimated to contain two or three protein molecules per asymmetric unit from the acceptable range of volume-to-weight ratio values.

  17. The effects of Alcea rosea L., Malva sylvestris L. and Salvia libanotica L. water extracts on the production of anti-egg albumin antibodies, interleukin-4, gamma interferon and interleukin-12 in BALB/c mice.

    PubMed

    El Ghaoui, Walid Bou Jaber; Ghanem, Elsa Bou; Chedid, Lara Abou; Abdelnoor, Alexander M

    2008-12-01

    Polysaccharides obtained from certain plants have been reported to have immunomodulatory properties. As a consequence of these reports the aim of this study was to investigate some immunomodulatory properties of water extracts of Alcea rosea L. (ARE), Malva sylvestris L. (MSE) and Salvia libanotica L. (SLE).Groups of egg albumin (EA)-immunized and -non-immunized Balb/c mice were treated with the carbohydrate-rich water extracts. Mice from each group were bled and their spleens removed at 3, 6 and 10 days post-immunization/treatment. Anti-egg albumin antibody levels in the processed sera were determined by an enzyme linked immunosorbent assay (ELISA). RNA was extracted from spleen cells and interleukin-4 (IL-4), interleukin-12 (IL-12) and gamma-interferon transcripts were determined by the reverse transcription polymerase chain reaction (RT-PCR).ARE appeared to boost the antibody response to EA, but had no effect on IL-4 and gamma-interferon gene transcription. MSE and SLE appeared to have no effect on anti-EA antibody production, but enhanced IL-12 and gamma-interferon gene transcription. MSE appeared to switch off, and SLE had no effect on, IL-4 transcription.In conclusion, it appears that ARE is a B-lymphocyte polyclonal activator, and MSE and SLE are macrophage and T helper-1 (Th-1) activators. PMID:18688815

  18. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

    PubMed

    Namadurai, Sivakumar; Jain, Deepti; Kulkarni, Dhananjay S; Tabib, Chaitanya R; Friedhoff, Peter; Rao, Desirazu N; Nair, Deepak T

    2010-01-01

    The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage. PMID:21060849

  19. Characterisation and stability of anthocyanins in purple-fleshed sweet potato P40.

    PubMed

    Xu, Jianteng; Su, Xiaoyu; Lim, Soyoung; Griffin, Jason; Carey, Edward; Katz, Benjamin; Tomich, John; Smith, J Scott; Wang, Weiqun

    2015-11-01

    Purple-fleshed sweet potato P40 has been shown to prevent colorectal cancer in a murine model. This study is to identify anthocyanins by using HPLC/MS-MS and assess the stability during various cooking conditions. P40 possesses a high content of anthocyanins up to 14 mg/g dry matter. Total 12 acylated anthocyanins are identified. Top three anthocyanins, e.g., cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside, peonidin 3-caffeoyl sophoroside-5-glucoside, and cyanidin 3-(6"-caffeoyl-6"-feruloylsophoroside)-5-glucoside, account for half of the anthocyanin contents. Over 80% of anthocyanins measured by acid hydrolysis were cyanidin derivatives, indicating P40 is unique when compared with other purple-fleshed sweet potatoes that usually contain more peonidin than cyanidin. Steaming, pressure cooking, microwaving, and frying but not baking significantly reduced 8-16% of total anthocyanin contents. Mono-acylated anthocyanins showed a higher resistance against heat than di- and non-acylated. Among of which, cyanidin 3-p-hydroxybenzoylsophoroside-5-glucoside exhibited the best thermal stability. The stable acylated and cyanidin-predominated anthocyanins in P40 may provide extra benefits for cancer prevention. PMID:25976796

  20. Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.

    PubMed

    Ha Lan, Thanh T; Chen, Stephanie J T; Arps, David P; Fullen, Douglas R; Patel, Rajiv M; Siddiqui, Javed; Carskadon, Shannon; Palanisamy, Nallasivam; Harms, Paul W

    2014-11-01

    Cutaneous spindle cell malignancies such as sarcomatoid squamous cell carcinoma (SCC), leiomyosarcoma, desmoplastic melanoma (DM) and atypical fibroxanthoma (AFX) may be morphologically indistinguishable, yet accurate diagnosis is important for appropriate clinical management. The distinction among these entities relies on immunohistochemical evaluation for epidermal, muscle or melanocytic differentiation. Epidermal differentiation markers include cytokeratins and p63. p63 is expressed as two distinct isoforms, ΔNp63 (p40) and TAp63. p40 positivity is highly specific for pulmonary SCC and head and neck sarcomatoid SCC. We examined the utility of p40 vs. p63 immunostaining in the differentiation of a variety of cutaneous spindle cell malignancies, including sarcomatoid SCC (n = 27), AFX (n = 34) and DM (n = 10). p40 was less sensitive than p63 for detecting sarcomatoid SCC (56% and 81%, respectively). p63 and p40 were comparably specific for sarcomatoid SCC relative to AFX, with only rare weak staining of tumor cells for p63 and/or p40 in a minority of AFX cases, including one case with approximately 10% of cells staining weakly for p40. All cases of DM were negative for p40 and p63. Our results support continued use of p63 for diagnosis of cutaneous sarcomatoid SCC because of greater sensitivity relative to p40. PMID:25263756

  1. The alkyl linkers in tandem-homodimers of a β-sheet-forming nonapeptide affect the self-assembled nanostructures.

    PubMed

    Tomizaki, Kin-Ya; Tanaka, Atsushi; Shimada, Hiroki; Nishizawa, Koki; Wada, Tsubasa; Imai, Takahito

    2016-06-01

    There is increasing interest in designing smart biomaterials by employing the self-assembly characteristics of synthetic peptides. The use of amyloid-like fibrils is one approach to nanometer- and micrometer-sized supramolecular structures. However, it is generally difficult to predict and/or analyze peptide conformations in nanostructures generated by the self-assembly of β-sheet-forming peptides such as amyloid-β peptide because each peptide experiences a slightly different environment. Therefore, a methodology for rationally designing peptide-based smart materials is required. In this study, we demonstrate the design and synthesis of tandem-homodimers of a β-sheet-forming peptide where the amino acid sequence is duplicated in series and joined via alkyl linkers of different chain length. The conformations of these tandem-homodimers within the self-assembled nanoarchitectures in aqueous solution were characterized. Our findings demonstrate that the hydrophobicity and/or flexibility of the alkyl linkers significantly affect the peptide conformation (extended or bent) of the self-assembled peptide nanostructures. We believe that the present tandem-homodimerization method represents a new direction for the rational design of peptide-based smart biomaterials. PMID:27117426

  2. Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4

    PubMed Central

    Nicoludis, John M; Vogt, Bennett E; Green, Anna G; Schärfe, Charlotta PI; Marks, Debora S; Gaudet, Rachelle

    2016-01-01

    Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families. DOI: http://dx.doi.org/10.7554/eLife.18449.001 PMID:27472898

  3. Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4.

    PubMed

    Nicoludis, John M; Vogt, Bennett E; Green, Anna G; Schärfe, Charlotta Pi; Marks, Debora S; Gaudet, Rachelle

    2016-01-01

    Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families. PMID:27472898

  4. Interleukin-12- and Gamma Interferon-Dependent Innate Immunity Are Essential and Sufficient for Long-Term Survival of Passively Immunized Mice Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Vollstedt, Sabine; Franchini, Marco; Alber, Gottfried; Ackermann, Mathias; Suter, Mark

    2001-01-01

    Interferon (IFN) type I (alpha/beta IFN [IFN-α/β]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-γ) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to104 PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-γ-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity. PMID:11559791

  5. Free IL-12p40 Monomer is a Polyfunctional Adapter for Generating Novel IL-12-Like Heterodimers Extracellularly

    PubMed Central

    Abdi, Kaveh; Singh, Nevil J.; Spooner, Eric; Kessler, Benedikt M.; Radaev, Sergei; Lantz, Larry; Xiao, Tsan Sam; Matzinger, Polly; Sun, Peter D.; Ploegh, Hidde L.

    2014-01-01

    IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23 respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. Here, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellulary with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners—the CD5 antigen-like glycoprotein CD5L— to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adapter which is able to generate multiple de novo composites in combination with other locally available polypeptide partners, post secretion. PMID:24821971

  6. Non-averaged human brain potentials in somatic attention: the short-latency cognition-related P40 component.

    PubMed Central

    Tomberg, C; Desmedt, J E

    1996-01-01

    1. Non-averaged scalp-recorded brain potentials were studied in humans during selective attention to randomly intermixed series of stimuli to fingers. Physiological tests were use for validating the presence or absence of the short-latency cognition-related P40 electrogeneses in parietal cortex in the response to a single-target stimulus (P40 signifies a positive polarity of about 40 ms peak latency). 2. To minimize interference from the electroencephalogram and noise we mapped single brain responses over the scalp and identified P40 topographies by an updated form of the numerical estimator Z for assessment of recorded potentials over time. We found that Z should exceed 0.96 for at least 15 ms for validation of the topographical congruity between the single P40 and an averaged P40 template. 3. Individual responses to 145 target finger stimuli correctly identified by the subject were analysed. P40 occurred only intermittently (34.5%) in a series of targets, but its voltage was unexpectedly large, exceeding the P40 voltage in averaged responses by a factor of about 10. 4. The usual assumption in the averaging method that the single brain responses combined in the average are stable but merely contaminated by unrelated noise was shown to be false for the cognition-related P40, which was considerably underestimated because of its intermittency in the averaged single trials. 5. The reaction time of the subject was on average 19% shorter in the trials in which a P40 was present, thus suggesting that P40 can influence subsequent perceptual processing by the brain in the same trial. 6. The feasibility of identifying specific cognition-related electrogeneses in single brain responses opens up the study of momentary shifts in brain processing strategies thereby allowing the neurophysiology of cognition to be based in real time. Images Figure 5 Figure 6 Figure 8 Figure 11 Figure 12 PMID:8910238

  7. A novel EID family member, EID-3, inhibits differentiation and forms a homodimer or heterodimer with EID-2

    SciTech Connect

    Sasajima, Yuka; Tanaka, Hiroyuki; Miyake, Satoshi; Yuasa, Yasuhito . E-mail: yuasa.monc@tmd.ac.jp

    2005-08-05

    The EID family members, i.e., E1A-like inhibitor of differentiation-1 (EID-1) and EID-1-like inhibitor of differentiation-2 (EID-2), were identified as negative regulators of cellular differentiation. EID-1 seems to inhibit differentiation by blocking histone acetyltransferase activity and EID-2 possibly inhibits differentiation through binding to class I histone deacetylases (HDACs). Here, we report a novel inhibitor of differentiation exhibiting homology with EID-2 termed EID-3 (EID-2-like inhibitor of differentiation-3). Like EID-2, EID-3 inhibited MyoD- and GR{alpha}-dependent transcription and blocked muscle differentiation in cultured cells by binding to class I HDACs. Unlike that of EID-2, the C-terminus, but not the N-terminus, of EID-3 was required for nuclear localization. EID-3 formed a homodimer or heterodimer with EID-2. These results suggest that EID-3 inhibits differentiation by blocking transcription as a complex in cells.

  8. Lactic Acid Bacteria Inducing a Weak Interleukin-12 and Tumor Necrosis Factor Alpha Response in Human Dendritic Cells Inhibit Strongly Stimulating Lactic Acid Bacteria but Act Synergistically with Gram-Negative Bacteria

    PubMed Central

    Zeuthen, Louise Hjerrild; Christensen, Hanne Risager; Frøkiær, Hanne

    2006-01-01

    The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G−) commensals, such as members of the family Enterobacteriaceae, may, however, be immunomodulators that are as important as G+ organisms but tend to be overlooked. Dendritic cells (DCs) are crucial immune regulators, and therefore, the present study aimed at investigating differences among human gut flora-derived LAB and G− bacteria in their patterns of DC polarization. Human monocyte-derived DCs were exposed to UV-killed bacteria, and cytokine secretion and surface marker expression were analyzed. Profound differences in the DC polarization patterns were found among the strains. While strains of LAB varied greatly in their capacity to induce interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α), G− strains were consistently weak IL-12 and TNF-α inducers. All strains induced significant amounts of IL-10, but G− bacteria were far more potent IL-10 inducers than LAB. Interestingly, we found that when weakly IL-12- and TNF-α-inducing LAB and strong IL-12- and TNF-α-inducing LAB were mixed, the weakly IL-12- and TNF-α-inducing LAB efficiently inhibited otherwise strong IL-12- and TNF-α-inducing LAB, yet when weakly IL-12- and TNF-α-inducing LAB were mixed with G− bacteria, they synergistically induced IL-12 and TNF-α. Furthermore, strong IL-12- and TNF-α-inducing LAB efficiently up-regulated surface markers (CD40, CD83, CD86, and HLA-DR), which were inhibited by weakly IL-12- and TNF-α-inducing LAB. All G− bacteria potently up-regulated surface markers; however, these markers were not inhibited by weakly IL-12- and TNF-α-inducing LAB. These much divergent DC stimulation patterns among intestinal bacteria, which encompass both antagonistic and synergistic relationships, support the

  9. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  10. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-04-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  11. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  12. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry.

    PubMed

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope. Graphical Abstract ᅟ. PMID:27067900

  13. Association of Common Genetic Polymorphisms with Melanoma Patient IL-12p40 Blood Levels, Risk, and Outcomes

    PubMed Central

    Fang, Shenying; Wang, Yuling; Chun, Yun S; Liu, Huey; Ross, Merrick I; Gershenwald, Jeffrey E; Cormier, Janice N; Royal, Richard E; Lucci, Anthony; Schacherer, Christopher W; Reveille, John D; Chen, Wei; Sui, Dawen; Bassett, Roland L; Wang, Li-E; Wei, Qingyi; Amos, Christopher I; Lee, Jeffrey E

    2015-01-01

    Recent investigation has identified association of IL-12p40 blood levels with melanoma recurrence and patient survival. No studies have investigated associations of single-nucleotide polymorphisms (SNPs) with melanoma patient IL-12p40 blood levels or their potential contributions to melanoma susceptibility or patient outcome. In the current study, 818,237 SNPs were available for 1,804 melanoma cases and 1,026 controls. IL-12p40 blood levels were assessed among 573 cases (discovery), 249 cases (case validation), and 299 controls (control validation). SNPs were evaluated for association with log[IL-12p40] levels in the discovery data set and replicated in two validation data sets, and significant SNPs were assessed for association with melanoma susceptibility and patient outcomes. The most significant SNP associated with log[IL-12p40] was in the IL-12B gene region (rs6897260, combined P=9.26 × 10−38); this single variant explained 13.1% of variability in log[IL-12p40]. The most significant SNP in EBF1 was rs6895454 (combined P=2.24 × 10−9). A marker in IL12B was associated with melanoma susceptibility (rs3213119, multivariate P=0.0499; OR=1.50, 95% CI 1.00–2.24), whereas a marker in EBF1 was associated with melanoma-specific survival in advanced-stage patients (rs10515789, multivariate P=0.02; HR=1.93, 95% CI 1.11–3.35). Both EBF1 and IL12B strongly regulate IL-12p40 blood levels, and IL-12p40 polymorphisms may contribute to melanoma susceptibility and influence patient outcome. PMID:25848976

  14. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    PubMed

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  15. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    PubMed Central

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  16. wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2

    PubMed Central

    Rideout, Marc C.; Naili, Ilham; Boldt, Jeffrey L.; Flores-Fujimoto, America; Patra, Sukanya; Rostron, Jason E.; Segall, Anca M.

    2013-01-01

    DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth. PMID:23291222

  17. IκBβ enhances the generation of the low-affinity NFκB/RelA homodimer

    PubMed Central

    Tsui, Rachel; Kearns, Jeffrey D.; Lynch, Candace; Vu, Don; Ngo, Kim; Basak, Soumen; Ghosh, Gourisankar; Hoffmann, Alexander

    2015-01-01

    The NFκB family of dimeric transcription factors regulate inflammatory and immune responses. While the dynamic control of NFκB dimer activity via the IκB-NFκB signaling module is well understood, there is little information on how specific dimer repertoires are generated from Rel family polypeptides. Here we report the iterative construction – guided by in vitro and in vivo experimentation – of a mathematical model of the Rel-NFκB generation module. Our study reveals that IκBβ has essential functions within the Rel-NFκB generation module, specifically for the RelA:RelA homodimer, which controls a subset of NFκB target genes. Our findings revise the current dogma of the three classical, functionally-related IκB proteins by distinguishing between a positive ‘licensing’ factor (IκBβ) that contributes to determining the available NFκB dimer repertoire in a cell’s steady state, and negative feedback regulators (IκBα and -ε) that determine the duration and dynamics of the cellular response to an inflammatory stimulus. PMID:25946967

  18. Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

    PubMed

    Yu, Hui-Chun; Lu, Ming-Chi; Huang, Kuang-Yung; Huang, Hsien-Lu; Liu, Su-Qin; Huang, Hsien-Bin; Lai, Ning-Sheng

    2016-01-01

    Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)₂ via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)₂ formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)₂ of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)₂ on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)₂ on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs. PMID:26729099

  19. Effect of p40tax trans-activator of human T cell lymphotropic virus type I on expression of autoantigens.

    PubMed

    Banki, K; Ablonczy, E; Nakamura, M; Perl, A

    1994-03-01

    The possibility of a retroviral etiology has long been raised in a number of autoimmune disorders. More recently, Sjögren's syndrome and rheumatoid arthritis were noted in transgenic mice carrying the tax gene of human T cell leukemia virus type I (HTLV-I). To evaluate the involvement of HTLV-I Tax in autoimmunity, its effect on expression of autoantigens was investigated. A metallothionein promoter-driven p40tax expression plasmid, pMAXRHneo-1, was stably transfected into Molt4 and Jurkat cells and the p40tax protein was induced with CdCl2. trans-Activation or trans-repression of autoantigens by HTLV-I Tax was studied by Western blot analysis utilizing autoantigen-specific murine monoclonal and rabbit polyvalent antibodies as well as sera from 161 autoimmune patients. Induction of p40tax of HTLV-I had no significant effect on levels of expression of common autoantigens U1 snRNP, Sm, Ro, La, HSP-70, topoisomerase I/Scl70, PCNA, and HRES-1. Expression of two potentially novel autoantigens, 44 and 46 kDa, was induced by p40tax as detected by sera of progressive systemic sclerosis patients, BAK and VAR. By contrast, expression of 24- and 34-kDa proteins was suppressed in response to induction of p40tax as detected by sera of systemic lupus erythematosus patients PUS and HOR. Because none of these patients were infected by HTLV-I, a protein functionally similar to p40tax may be involved in eliciting autoantigen expression and a subsequent autoantibody response in a minority of patients with PSS and SLE. Sera of autoimmune patients may also be utilized to detect novel proteins trans-activated or trans-repressed by p40tax of HTLV-I. PMID:8018391

  20. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer.

    PubMed

    Shen, Huaishun; Cao, Kaiming; Wang, Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors. PMID:17719007

  1. Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Thondorf, Iris; Theisgen, Stephan; Lilie, Hauke; Schröder, Thomas; Arlt, Christian; Ihling, Christian H.; Sinz, Andrea

    2013-12-01

    The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

  2. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer

    SciTech Connect

    Shen Huaishun; Cao Kaiming; Wang Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

  3. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    SciTech Connect

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  4. Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

    PubMed Central

    Thoden, J. B.; Holden, H. M.; Fisher, A. J.; Sinclair, J. F.; Wesenberg, G.; Baldwin, T. O.; Rayment, I.

    1997-01-01

    Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas. PMID:9007973

  5. Structural Basis for a Munc13–1 Homodimer to Munc13–1/RIM Heterodimer Switch

    PubMed Central

    Lu, Jun; Machius, Mischa; Dulubova, Irina; Dai, Han; Südhof, Thomas C; Tomchick, Diana R

    2006-01-01

    C 2 domains are well characterized as Ca 2+/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C 2A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C 2A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C 2A-domain homodimer and the Munc13–1 C 2A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C 2A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C 2A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C 2 domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes. PMID:16732694

  6. Substrate-Modulated Thermal Fluctuations Affect Long-Range Allosteric Signaling in Protein Homodimers: Exemplified in CAP

    PubMed Central

    Toncrova, Hedvika; McLeish, Tom C.B.

    2010-01-01

    Abstract The role of conformational dynamics in allosteric signaling of proteins is increasingly recognized as an important and subtle aspect of this ubiquitous phenomenon. Cooperative binding is commonly observed in proteins with twofold symmetry that bind two identical ligands. We construct a coarse-grained model of an allosteric coupled dimer and show how the signal can be propagated between the distant binding sites via change in slow global vibrational modes alone. We demonstrate that modulation on substrate binding of as few as 5–10 slow modes can give rise to cooperativity observed in biological systems and that the type of cooperativity is given by change of interaction between the two monomers upon ligand binding. To illustrate the application of the model, we apply it to a challenging test case: the catabolite activator protein (CAP). CAP displays negative cooperativity upon association with two identical ligands. The conformation of CAP is not affected by the binding, but its vibrational spectrum undergoes a strong modification. Intriguingly, the first binding enhances thermal fluctuations, yet the second quenches them. We show that this counterintuitive behavior is, in fact, necessary for an optimal anticooperative system, and captured within a well-defined region of the model's parameter space. From analyzing the experimental results, we conclude that fast local modes take an active part in the allostery of CAP, coupled to the more-global slow modes. By including them into the model, we elucidate the role of the modes on different timescales. We conclude that such dynamic control of allostery in homodimers may be a general phenomenon and that our model framework can be used for extended interpretation of thermodynamic parameters in other systems. PMID:20483341

  7. The enzyme engineering of mutant homodimer and heterodimer of coproporphyinogen oxidase contributes to new insight into hereditary coproporphyria and harderoporphyria.

    PubMed

    Kim, Dao Hoang Thien; Hino, Ryoko; Adachi, Yuka; Kobori, Akio; Taketani, Shigeru

    2013-12-01

    Hereditary coproporphyria (HCP) is an autosomal dominant-inherited disease of haem biosynthesis caused by partial deficiency of the enzyme coproporphyrinogen oxidase (CPOX). Patients with HCP show <50% of normal activity and those with the rare autosomal recessive harderoporphyria accumulate harderoporphyrinogen, an intermediate porphyrin of the CPOX reaction. To clarify the relationship of the low enzyme activity with these diseases, we expressed mutant CPOX carrying His-tag from these porphyria patients and co-expressed mutant CPOX carrying His-tag and normal CPOX carrying HA-tag in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms a heterodimer in association with the HA-enzyme, and analysis using a cross-link reagent confirmed that the enzyme is a dimer (∼70 kDa). Then, we expressed homo- and heterodimers composed of the wild-type (wt) and engineered mutants of the enzyme or mutants from HCP patients. The monomer form of mutated CPOX did not show any activity and homodimeric enzymes derived from HCP mutant showed low activity (<20% of the control). Some mutations of amino acids 401-404 were associated with marked accumulation of harderoporphyrinogen, with a decrease in the production of protoporphyrinogen, whereas K404E derived from patients with harderoporphyria produced less harderoporphyrinogen. The heterodimers with wt and mutated subunits from HCP patients showed low protoporphyrinogen producing activity. These results show that the substitution of amino acids from R401 to K404 results in extremely low enzyme activity with either mutant homodimer or heterodimers containing normal and mutated subunits and can be linked to HCP disease. PMID:24078084

  8. On a fully closed state of native human type-1 VDAC enriched in Nonidet P40.

    PubMed

    Thinnes, Friedrich P; Burckhardt, Gerhard

    2012-11-01

    There is indication that human type-1 VDAC/Porin31HL complexes, when purified from highly enriched cell membrane preparations of human B-lymphocytes by classical ion-exchange chromatography in the detergent Nonidet P40, rest in fully closed state, its N-terminus being accessible for mAbs. Cholesterol appears to be involved as a channel modulator. The channel switches to anion-selective or "open state" while being incorporated into black membranes at zero transmembrane potential. In this case, its N-terminus is hidden in the channel lumen. The cation-selective or "closed state" can be induced by transmembrane potentials beyond 30 mV, the N-terminus putatively now being positioned outside the channel lumen. The latter situation might allow one to decide if type-1 VDAC, preincubated with adequate antibodies against its N-terminal part, would enter black membranes in fully closed state or stay in the application medium, respectively, may be complexed to dimers. PMID:23000107

  9. Regulation of alveolar macrophage p40phox: hierarchy of activating kinases and their inhibition by PGE2

    PubMed Central

    Bourdonnay, Emilie; Serezani, Carlos H.; Aronoff, David M.; Peters-Golden, Marc

    2012-01-01

    PGE2, produced in the lung during infection with microbes such as Klebsiella pneumoniae, inhibits alveolar macrophage (AM) antimicrobial functions by preventing H2O2 production by NADPH oxidase (NADPHox). Activation of the NADPHox complex is poorly understood in AMs, although in neutrophils it is known to be mediated by kinases including PI3K/Akt, protein kinase C (PKC) δ, p21-activated protein kinase (PAK), casein kinase 2 (CK2), and MAPKs. The p40phox cytosolic subunit of NADPHox has been recently recognized to function as a carrier protein for other subunits and a positive regulator of oxidase activation, a role previously considered unique to another subunit, p47phox. The regulation of p40phox remains poorly understood, and the effect of PGE2 on its activation is completely undefined. We addressed these issues in rat AMs activated with IgG-opsonized K. pneumoniae. The kinetics of kinase activation and the consequences of kinase inhibition and silencing revealed a critical role for a PKCδ-PAK-class I PI3K/Akt1 cascade in the regulation of p40phox activation upon bacterial challenge in AMs; PKCα, ERK, and CK2 were not involved. PGE2 inhibited the activation of p40phox, and its effects were mediated by protein kinase A type II, were independent of interactions with anchoring proteins, and were directed at the distal class I PI3K/Akt1 activation step. Defining the kinases that control AM p40phox activation and that are the targets for inhibition by PGE2 provides new insights into immunoregulation in the infected lung. PMID:22544939

  10. Suppression of IL-12p40-related regulatory cytokines by suberoylanilide hydroxamic acid an inhibitor of histone deacetylases.

    PubMed

    Dobreva, Zlatka Georgieva; Grigorov, Boncho Grigorov; Stanilova, Spaska Angelova

    2016-08-01

    Small molecule inhibitors of histone deacetylases (HDACs) are a new class drugs used in clinical trials for the treatment of various malignancies. Emerging evidence suggest that HDAC inhibitors may also have anti-inflammatory properties, although the molecular mechanisms remain poorly defined. Our study investigates the effect of the HDACs inhibitor suberoylanilide hydroxamic acid (SAHA) on the expression of IL-12p40-related cytokines. For this purpose, human peripheral blood mononuclear cells (PBMC) were stimulated with LPS and C3bgp with or without SAHA. IL-12p40, IL-12p35 and IL-23p19 mRNA was determined at 6 h by qRT-PCR. Cytokine levels were determined in culture supernatants at 6 and 24 h, by ELISA. SAHA significantly inhibited IL-12p40 and IL-23p19 mRNA synthesis and did not change IL-12p35 mRNA transcription. Early at 6 h, we detected significantly decreased IL-12p40 and IL-23, but not IL-12p70 protein production in cultures treated with SAHA. Results also showed that the suppression of IL-12p40-related cytokines was clearly defined at 24 h. However, this suppression was less pronounced regarding IL-12p70. The present study showed that SAHA suppressed the gene expression of IL-23p19 stronger than the expression of IL-12p35, as well as the synthesis of IL-23 compared to that of IL-12p70. We suggest that this inhibitory effect of SAHA may be beneficial during treatment of inflammatory and autoimmune diseases mediated by Th17 immune response. PMID:27240992

  11. Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer

    PubMed Central

    2010-01-01

    Background We have recently described the correlation between quantitative measures of HER2 expression or HER2 homodimers by the HERmark assay and objective response (RR), time-to progression (TTP), and overall survival (OS) in an expanded access cohort of trastuzumab-treated HER2-positive patients with metastatic breast cancer (MBC) who were stringently selected by fluorescence in situ hybridization (FISH). Multivariate analyses suggested a continuum of HER2 expression that correlated with outcome following trastuzumab. Here we investigate the relationship between HER2 expression or HER2 homodimers and OS in a clinic-based population of patients with MBC selected primarily by IHC. Methods HERmark, a proximity-based assay designed to detect and quantitate protein expression and dimerization in formalin-fixed paraffin-embedded (FFPE) tissues, was used to measure HER2 expression and HER2 homodimers in FFPE samples from patients with MBC. Assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots, and multivariate Cox regression analyses. Results Initial analyses revealed a parabolic relationship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of linearity for the HER2 expression measurements may be inappropriate in subsequent multivariate analyses. Cox regression analyses using the categorized variable of HER2 expression level demonstrated that higher HER2 levels predicted better survival outcomes following trastuzumab treatment in the high HER2-expressing group. Conclusions These data suggest that the quantitative amount of HER2 expression measured by Hermark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with MBC. PMID:20178580

  12. Assembly of the Bak apoptotic pore: a critical role for the Bak protein α6 helix in the multimerization of homodimers during apoptosis.

    PubMed

    Ma, Stephen; Hockings, Colin; Anwari, Khatira; Kratina, Tobias; Fennell, Stephanie; Lazarou, Michael; Ryan, Michael T; Kluck, Ruth M; Dewson, Grant

    2013-09-01

    Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins. PMID:23893415

  13. Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions*

    PubMed Central

    Lin, Wei; Wang, Ying; Han, Xiaobiao; Zhang, Zilong; Wang, Chengyuan; Wang, Jin; Yang, Huaiyu; Lu, Yinhua; Jiang, Weihong; Zhao, Guo-Ping; Zhang, Peng

    2014-01-01

    The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp50 residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg52 from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp50 with the highly conserved Arg52 and Thr9 in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation. PMID:24733389

  14. High Brightness Picture Technology In SD-P40 Projection TV

    NASA Astrophysics Data System (ADS)

    Hasegawa, Shinichi

    1987-04-01

    Pioneer Electric Company has developed a new generation 40-inch rear projection SD-P40 television, a revolutionary, new television that realizes white peak brightness of 300 ft-I and high contrast. The combination of high brightness and high contrast is made possible primarily by newly developed optical-coupling technology that utilizes newly developed concepts. This new optical coupling technology cools the CRT quite efficiently, making it possible to greatly increase the CRT power input to obtain high brightness and at the same time provides greater reliability than direct view televisions. The new optical-coupling technology also makes it possible to almost completely eliminate the reflectance at the boundaries between the CRT and the lens and air, which gives much higher contrast than previous televisions. Not only does this optical-coupling technology provide high performance, in addition since the liquid coolant it employs functions as a liquid lens, the coupling lens can be designed to a uniform thinness and a small aperture. This greatly reduces the cost of the lens. Our newly developed optical-coupling technology is the ultimate form of cooling for the CRT tubes of projection televisions and coupling with the lens and will become the mainstream technology in the future. It is forecast that other manufacturers will also adopt this type of technology. The optical lens section, which is the heart of a projection television, is a hybrid structure with three aspherical plastic lenses and one glass spherical lens. It has higher performance image formation and greater temperature stability than previous televisions. The plastic lenses are all finished with multi-coating to hold down light loss and maximize transparency. This con-tributes greatly to increasing the brightness for a projection television. Previous 3-tube type projection televisions were bothered by low color uniformity, color shift, and low color rela-tive illumination. This model uses three bends

  15. Subcellular localisation of the p40phox component of NADPH oxidase involves direct interactions between the Phox homology domain and F-actin

    PubMed Central

    Shao, Dongmin; Segal, Anthony W.; Dekker, Lodewijk V.

    2010-01-01

    Cytosolic components of the NADPH oxidase interact with the actin cytoskeleton. These interactions are thought to be important for the activation of this enzyme system but they are poorly characterised at the molecular level. Here we have explored the interaction between the actin cytoskeleton and p40phox, one of the cytosolic components of NADPH oxidase. Full length p40phox expressed in COS cells co-localised with F-actin in a peripheral lamellar compartment. The co-localisation was lost after deletion of the Phox homology (PX) domain and the PX domain in isolation (p40PX) showed the same F-actin co-localisation as the full length protein. PX domains are known lipid-binding modules however, a mutant p40PX which did not bind lipids still co-localised with F-actin suggesting that lipid-independent interactions underlie the localisation. Affinity chromatography identified actin as a binding partner for p40PX in neutrophil extracts. Pure actin interacted with both p40phox and with p40PX suggesting it is a direct interaction. Disruption of the actin cytoskeleton with cytochalasin D resulted in actin rearrangement and concomitantly the localisation of full length p40phox proteins and that of p40PX changed. Thus p40PX is a dual F-actin/lipid-binding module and F-actin interactions with the PX domain dictate at least in part the intracellular localisation of the cytosolic p40phox subunit of the NADPH oxidase. PMID:20637895

  16. A Lactobacillus rhamnosus GG-derived Soluble Protein, p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor*

    PubMed Central

    Yan, Fang; Liu, Liping; Dempsey, Peter J.; Tsai, Yu-Hwai; Raines, Elaine W.; Wilson, Carole L.; Cao, Hailong; Cao, Zheng; Liu, LinShu; Polk, D. Brent

    2013-01-01

    p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17−/− MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17−/− MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR. PMID:24043629

  17. Microbiota downregulates dendritic cell expression of miR-10a, which targets IL-12/IL-23p40.

    PubMed

    Xue, Xiaochang; Feng, Ting; Yao, Suxia; Wolf, Kyle J; Liu, Chang-Gong; Liu, Xiuping; Elson, Charles O; Cong, Yingzi

    2011-12-01

    Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases. The microRNAs (miRNAs), a class of small, noncoding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells, as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of inflammatory bowel disease. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, because the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free mice, expressed much lower levels of miR-10a compared with those in germ-free mice. Commensal bacteria downregulated dendritic cell miR-10a expression via TLR-TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited, whereas the miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in dendritic cells. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibited lower levels of intestinal miR-10a compared with control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression. PMID:22068236

  18. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  19. FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

    PubMed Central

    Tian, Wei; Li, Xing Jun; Stull, Natalie D.; Ming, Wenyu; Suh, Chang-Il; Bissonnette, Sarah A.; Yaffe, Michael B.; Grinstein, Sergio; Atkinson, Simon J.

    2008-01-01

    The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. PMID:18711001

  20. Neutrophil elastase enhances IL-12p40 production by lipopolysaccharide-stimulated macrophages via transactivation of the PAR-2/EGFR/TLR4 signaling pathway.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-07-01

    Proteinase-activated receptor 2 (PAR-2) and toll-like receptor 4 (TLR4) are involved in innate immune responses and signaling cross-talk between these receptor molecules has the potential to augment an ongoing inflammatory response. The aim of this study was to evaluate the possible cooperative influence of PAR-2 and TLR4 on IL-12p40 production by macrophages after stimulation with lipopolysaccharide (LPS). During culture, GM-CSF upregulated PAR-2 expression by macrophages in a time-dependent manner. Stimulation with LPS enhanced IL-12p40 production by macrophages in a concentration-dependent manner. While human neutrophil elastase (HNE) did not induce IL-12p40 production, pretreatment of macrophages with HNE synergistically increased the IL-12p40 protein level after LPS exposure. Silencing of TLR4 with small interfering RNA blunted the synergistic enhancement of IL-12p40 by HNE combined with LPS. Silencing of β-arrestin 2, p22phox, or ERK1/2 also inhibited an increase of IL-12p40. Interestingly, transfection of macrophages with small interfering RNA duplexes for DUOX-2, EGFR, TLR4, or TRAF6 significantly blunted the increase of IL-12p40 in response to treatment with HNE plus LPS. U73122 and Rottlerin also inhibited the increased production of IL-12p40. In conclusion, HNE is involved in transactivation of TLR4 through activation of DUOX-2/EGFR and synergistically enhances IL-12p40 production by macrophages stimulated with LPS. PMID:27282560

  1. An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers.

    PubMed

    Lensing, Cody J; Freeman, Katie T; Schnell, Sathya M; Adank, Danielle N; Speth, Robert C; Haskell-Luevano, Carrie

    2016-04-14

    Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects. PMID:26959173

  2. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

    PubMed Central

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates. PMID:25658443

  3. HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

    PubMed Central

    Marroquin Belaunzaran, Osiris; Kleber, Sascha; Schauer, Stefan; Hausmann, Martin; Nicholls, Flora; Van den Broek, Maries; Payeli, Sravan; Ciurea, Adrian; Milling, Simon; Stenner, Frank; Shaw, Jackie; Kollnberger, Simon; Bowness, Paul; Petrausch, Ulf; Renner, Christoph

    2015-01-01

    Objectives HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. Methods The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. Results HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. Conclusion HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders. PMID:26125554

  4. Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells

    PubMed Central

    Yu, Chao; Crispin, Max; Sonnen, Andreas F.-P.; Harvey, David J.; Chang, Veronica T.; Evans, Edward J.; Scanlan, Christopher N.; Stuart, David I.; Gilbert, Robert J. C.; Davis, Simon J.

    2011-01-01

    Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3–5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3–4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a = 43.9, b = 51.5, c = 102.9 Å and space group P212121 that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems. PMID:21795794

  5. Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

    PubMed

    Gherbi, Karolina; May, Lauren T; Baker, Jillian G; Briddon, Stephen J; Hill, Stephen J

    2015-07-01

    At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min(-1) in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation. PMID:25837585

  6. Carcinogenic heavy metals replace Ca{sup 2+} for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer

    SciTech Connect

    Hirata, Aiko; Corcoran, George B.; Hirata, Fusao

    2010-10-01

    Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg{sup 2+}- and ATP-dependent manner, and to anneal ssDNA in a Ca{sup 2+}-dependent manner. Phospholipids decreased the concentration of Ca{sup 2+} required for maximal annealing activity. Heavy metals such as As{sup 3+}, Cr{sup 6+}, Pb{sup 2+} and Cd{sup 2+} substituted for Ca{sup 2+} in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg{sup 2+} and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/-) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As{sup 3+}- or Cr{sup 6+}-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As{sup 3+} or Cr{sup 6+} towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

  7. Next Step toward Optimization of GRP Receptor Avidities: Determination of the Minimal Distance between BBN(7-14) Units in Peptide Homodimers.

    PubMed

    Fischer, G; Lindner, S; Litau, S; Schirrmacher, R; Wängler, B; Wängler, C

    2015-08-19

    As the gastrin releasing peptide receptor (GRPR) is overexpressed on several tumor types, it represents a promising target for the specific in vivo imaging of these tumors using positron emission tomography (PET). We were able to show that PESIN-based peptide multimers can result in substantially higher GRPR avidities, highly advantageous in vivo pharmacokinetics and tumor imaging properties compared to the respective monomers. However, the minimal distance between the peptidic binders, resulting in the lowest possible system entropy while enabling a concomitant GRPR binding and thus optimized receptor avidities, has not been determined so far. Thus, we aimed here to identify the minimal distance between two GRPR-binding peptides in order to provide the basis for the development of highly avid GRPR-specific PET imaging agents. We therefore synthesized dimers of the GRPR-binding bombesin analogue BBN(7-14) on a dendritic scaffold, exhibiting different distances between both peptide binders. The homodimers were further modified with the chelator NODAGA, radiolabeled with (68)Ga, and evaluated in vitro regarding their GRPR avidity. We found that the most potent of the newly developed radioligands exhibits GRPR avidity twice as high as the most potent reference compound known so far, and that a minimal distance of 62 bond lengths between both peptidic binders within the homodimer can result in concomitant peptide binding and optimal GRPR avidities. These findings answer the question as to what molecular design should be chosen when aiming at the development of highly avid homobivalent peptidic ligands addressing the GRPR. PMID:26200324

  8. p40 is the best marker for diagnosing pulmonary squamous cell carcinoma: comparison with p63, cytokeratin 5/6, desmocollin-3, and sox2.

    PubMed

    Tatsumori, Takahiro; Tsuta, Koji; Masai, Kyohei; Kinno, Tomoaki; Taniyama, Tomoko; Yoshida, Akihiko; Suzuki, Kenji; Tsuda, Hitoshi

    2014-01-01

    Histologic distinction among non-small cell lung carcinomas, particularly between squamous cell carcinoma (SQC) and adenocarcinoma (ADC), has become more important. Recently, a p40 antibody was suggested to be a highly specific marker for SQC. We evaluated p40 expression and compared it with the expression of other SQC markers in 580 primary lung carcinomas, including 158 SQCs, 156 ADCs, 50 carcinoid tomors, 107 large cell neuroendocrine carcinomas, 68 small cell lung carcinomas, and 41 malignant mesotheliomas. Detailed histologic distributions of p40-positive cases were as follows: 153 (96.8%) of 158 SQCs, 7 (4.6%) of 152 ADCs, 0 (0%) of 50 carcinoid tomors, 4 (3.6%) of 107 large cell neuroendocrine carcinomas, 1 (1.5%) of 68 small cell lung carcinomas, and 1 (2.4%) of 41 mesotheliomas. p40 staining yields high sensitivity as well as high specificity for distinguishing SQC from ADC, neuroendocrine carcinomas, and malignant mesothelioma. PMID:24805133

  9. Comparison of p63 and p40 (ΔNp63) as Basal, Squamoid, and Myoepithelial Markers in Salivary Gland Tumors.

    PubMed

    Owosho, Adepitan A; Aguilar, Cristina E; Seethala, Raja R

    2016-08-01

    p40 is selective for ΔNp63 isoforms and appears to be more specific for squamous differentiation than p63. Its performance as a basal/myoepithelial marker in salivary gland tumors has only rarely been addressed in the literature. We thus compared the performance of p63 and p40 (ΔNp63) immunohistochemical stain as markers of basal, squamoid, and myoepithelial differentiation in 105 salivary gland tumors selected from our archives. The neoplasms were categorized according to their presumed phenotype as ductoacinar (n=45), biphasic (dual ductal and myoepithelial/basal differentiation, n=44), purely myoepithelial (n=5), and excretory duct phenotype (n=11). Only nuclear staining for p63 and p40 was considered positive. Distribution of staining was scored as: 0 (no staining), 1+ (1% to 25%), 2+ (26% to 50%), 3+ (51% to 75%), and 4+ (76% to 100%). Intensity was scored as weak, moderate, or strong. p63 and p40 highlighted the basal and myoepithelial cells in normal salivary gland tissue as well as basal/myoepithelial/squamoid elements in biphasic tumors, purely myoepithelial tumors, and excretory duct type tumors (4+ with strong staining for p63, and moderate staining for p40). All ductal tumors were negative for p40. However, 13/13 polymorphous low-grade adenocarcinoma/cribriform adenocarcinomas of salivary gland, 7/9 canalicular adenomas, and 3/5 mammary analog secretory carcinomas showed some degree of p63 staining. Thus, we confirm that p40 is a more specific basal/myoepithelial/squamoid marker than p63 in salivary gland tumors. A subset of ductal tumors show a discordant p63+/p40- immunoprofile that can be a pitfall if not recognized, but may also help distinguish these tumors from truly biphasic tumors and myoepithelial tumors. PMID:26230372

  10. Identification and immuno-electron microscopy localization of p40, a protein component of immunosuppressive virus-like particles from Leptopilina heterotoma, a virulent parasitoid wasp of Drosophila.

    PubMed

    Chiu, Hsiling; Morales, Jorge; Govind, Shubha

    2006-02-01

    Lamellocytes are specialized larval blood cells of Drosophila that carry out encapsulation of metazoan pathogens such as parasitoid wasps. Large virus-like particles (VLPs) from two closely related virulent parasitoid wasp species, Leptopilina heterotoma and Leptopilina victoriae, suppress the host encapsulation response by promoting lysis of lamellocytes. The molecular basis of VLP-lamellocyte interaction and lamellocyte lysis is not understood. Here, it was shown that mature VLPs are composed of at least four major proteins. Polyclonal antisera against the most abundant L. heterotoma VLP protein, p40, cross-reacted with the most abundant L. victoriae VLP protein, p47.5. Immuno-electron microscopy (EM) of the long gland-reservoir complex revealed that p40 was expressed early in VLP biogenesis and was detected along with VLP precursors within the long gland cells and lumen. In the reservoir, VLPs had an angular core, resembled mature particles and p40 was detected outside the VLP cores. Immuno-EM staining of mature VLPs from both species localized the p40 and p47.5 proteins largely to the periphery of the VLPs and along the VLP spike-like projections. p40 staining was observed in VLP-treated host haemocytes. In vitro, anti-p40 antibody almost completely blocked the ability of L. heterotoma VLPs to promote lamellocyte lysis. Anti-p40 antibody blocked lysis by L. victoriae VLPs by >50%. It is proposed that the VLP surface proteins p40 and p47.5 share antigenic determinants and significantly contribute to the strong virulence of their Hymenopteran hosts. PMID:16432035

  11. Herpes simplex virus type 1 and 2 intracellular p40: type-specific and cross-reactive antigenic determinants on peptides generated by partial proteolysis.

    PubMed Central

    Heilman, C J; Zweig, M; Hampar, B

    1981-01-01

    Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants. Images PMID:6172597

  12. Single Nucleotide Polymorphisms in IL-10, IL-12p40, and IL-13 Genes and Susceptibility to Glioma

    PubMed Central

    Shamran, Haidar A.; Ghazi, Haidar F.; AL-Salman, Ahmed; Al-Juboory, Ahmad A.; Taub, Dennis D.; Price, Robert L.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.; Singh, Udai P.

    2015-01-01

    Glioma is one of the most aggressive and most common tumors of the central nervous system (CNS) in humans. The exact causes of glioma are not well known, but evidence suggests the involvement of genetic factors in addition to environmental risk factors. The present study aimed to determine whether polymorphisms in IL-10-1082A/G, IL-12p40 1188C/A, and IL-13+2044G/A (rs20541) are associated with the incidence of glioma in Iraqi patients. Ninety-six patients with different grades of glioma and 40 apparently healthy individuals were recruited. A blood sample and genomic DNA were collected from all subjects. The amplification refractory mutation system and sequence-specific primer polymerase chain reaction (PCR) were used for genotyping of IL-10-1082A/G and IL-12p40 1188C/A, respectively; whereas, the IL-13+2044G/A was detected by DNA sequencing after amplification of the genes by PCR. All SNPs were within Hardy-Weinberg equilibrium and each appeared in three genotypes in patients and controls. In IL-10-1082A/G, these genotypes frequencies were AA (75%), AG (22.93%) and GG (2.07%) in patients as compared to similar frequencies (62.5%), (27.5%) and (10%) respectively, in controls. The variant IL-12p40 1188C/A genotype was AA (72.92%), AC (23.96%), and CC (3.13%%) in patients as compared to 65%, 30%, and 5%, respectively, in controls. The frequencies of IL-13+2044G/A genotypes (GG, GA, and AA) were 89.58%, 9.37%, and 1.04% among patients versus 47.5%, 32.5% and 20%, respectively, among controls. These results suggest a protective role of mutant alleles G and A in IL-10-1082A/G and IL-13+2044G/A against gliomas. Further studies with more rigorous parameter designs will be needed to confirm the current findings. PMID:26516307

  13. Proton-bound dimers of nitrogen heterocyclic molecules: Substituent effects on the structures and binding energies of homodimers of diazine, triazine, and fluoropyridine

    SciTech Connect

    Attah, Isaac K.; Platt, Sean P.; Meot-Ner, Michael; El-Shall, M. S.; Aziz, Saadullah G.; Alyoubi, Abdulrahman O.

    2014-03-21

    The bonding energies of proton-bound homodimers BH{sup +}B were measured by ion mobility equilibrium studies and calculated at the DFT B3LYP/6-311++G{sup **} level, for a series of nitrogen heterocyclic molecules (B) with electron-withdrawing in-ring N and on-ring F substituents. The binding energies (ΔH°{sub dissoc}) of the proton-bound dimers (BH{sup +}B) vary significantly, from 29.7 to 18.1 kcal/mol, decreasing linearly with decreasing the proton affinity of the monomer (B). This trend differs significantly from the constant binding energies of most homodimers of other organic nitrogen and oxygen bases. The experimentally measured ΔH°{sub dissoc} for (1,3-diazine){sub 2}H{sup +}, i.e., (pyrimidine){sub 2}H{sup +} and (3-F-pyridine){sub 2}H{sup +} are 22.7 and 23.0 kcal/mol, respectively. The measured ΔH°{sub dissoc} for the pyrimidine{sup ·+}(3-F-pyridine) radical cation dimer (19.2 kcal/mol) is signifcantly lower than that of the proton-bound homodimers of pyrimidine and 3-F-pyridine, reflecting the stronger interaction in the ionic H-bond of the protonated dimers. The calculated binding energies for (1,2-diazine){sub 2}H{sup +}, (pyridine){sub 2}H{sup +}, (2-F-pyridine){sub 2}H{sup +}, (3-F-pyridine){sub 2}H{sup +}, (2,6-di-F-pyridine){sub 2}H{sup +}, (4-F-pyridine){sub 2}H{sup +}, (1,3-diazine){sub 2}H{sup +}, (1,4-diazine){sub 2}H{sup +}, (1,3,5-triazine){sub 2}H{sup +}, and (pentafluoropyridine){sub 2}H{sup +} are 29.7, 24.9, 24.8, 23.3, 23.2, 23.0, 22.4, 21.9, 19.3, and 18.1 kcal/mol, respectively. The electron-withdrawing substituents form internal dipoles whose electrostatic interactions contribute to both the decreased proton affinities of (B) and the decreased binding energies of the protonated dimers BH{sup +}B. The bonding energies also vary with rotation about the hydrogen bond, and they decrease in rotamers where the internal dipoles of the components are aligned efficiently for inter-ring repulsion. For compounds substituted at the 3 or 4

  14. Proton-bound dimers of nitrogen heterocyclic molecules: Substituent effects on the structures and binding energies of homodimers of diazine, triazine, and fluoropyridine

    NASA Astrophysics Data System (ADS)

    Attah, Isaac K.; Platt, Sean P.; Meot-Ner Mautner, Michael; El-Shall, M. S.; Aziz, Saadullah G.; Alyoubi, Abdulrahman O.

    2014-03-01

    The bonding energies of proton-bound homodimers BH+B were measured by ion mobility equilibrium studies and calculated at the DFT B3LYP/6-311++G** level, for a series of nitrogen heterocyclic molecules (B) with electron-withdrawing in-ring N and on-ring F substituents. The binding energies (ΔH°dissoc) of the proton-bound dimers (BH+B) vary significantly, from 29.7 to 18.1 kcal/mol, decreasing linearly with decreasing the proton affinity of the monomer (B). This trend differs significantly from the constant binding energies of most homodimers of other organic nitrogen and oxygen bases. The experimentally measured ΔH°dissoc for (1,3-diazine)2H+, i.e., (pyrimidine)2H+ and (3-F-pyridine)2H+ are 22.7 and 23.0 kcal/mol, respectively. The measured ΔH°dissoc for the pyrimidine.+(3-F-pyridine) radical cation dimer (19.2 kcal/mol) is signifcantly lower than that of the proton-bound homodimers of pyrimidine and 3-F-pyridine, reflecting the stronger interaction in the ionic H-bond of the protonated dimers. The calculated binding energies for (1,2-diazine)2H+, (pyridine)2H+, (2-F-pyridine)2H+, (3-F-pyridine)2H+, (2,6-di-F-pyridine)2H+, (4-F-pyridine)2H+, (1,3-diazine)2H+, (1,4-diazine)2H+, (1,3,5-triazine)2H+, and (pentafluoropyridine)2H+ are 29.7, 24.9, 24.8, 23.3, 23.2, 23.0, 22.4, 21.9, 19.3, and 18.1 kcal/mol, respectively. The electron-withdrawing substituents form internal dipoles whose electrostatic interactions contribute to both the decreased proton affinities of (B) and the decreased binding energies of the protonated dimers BH+B. The bonding energies also vary with rotation about the hydrogen bond, and they decrease in rotamers where the internal dipoles of the components are aligned efficiently for inter-ring repulsion. For compounds substituted at the 3 or 4 (meta or para) positions, the lowest energy rotamers are T-shaped with the planes of the two rings rotated by 90° about the hydrogen bond, while the planar rotamers are weakened by repulsion between the

  15. Localized interleukin-12 delivery for immunotherapy of solid tumours.

    PubMed

    Wei, Louis Z; Xu, Yixin; Nelles, E Megan; Furlonger, Caren; Wang, James C M; Di Grappa, Marco A; Khokha, Rama; Medin, Jeffrey A; Paige, Christopher J

    2013-11-01

    Interleukin (IL)-12 is the key cytokine in the initiation of a Th1 response and has shown promise as an anti-cancer agent; however, clinical trials involving IL-12 have been unsuccessful due to toxic side-effects. To address this issue, lentiviral vectors were used to transduce tumour cell lines that were injected as an autologous tumour cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumour model. SCCVII cells that were transduced to produce IL-12 at different concentrations were then isolated. Subcutaneous injection of parental SCCVII cells results in tumour development, while a mixture of IL-12-producing and non-producing cells results in tumour clearance. Interestingly, when comparing mice injected a mixture of SCCVII and either high IL-12-producing tumour cells or low IL-12-producing tumour cells, we observed that mixtures containing small amounts of high producing cells lead to tumour clearance, whereas mixtures containing large amounts of low producing cells fail to elicit protection, despite the production of equal amounts of total IL-12 in both mixtures. Furthermore, immunizing mice with IL-12-producing cells leads to the establishment of both local and systemic immunity against challenge with SCCVII. Using depletion antibodies, it was shown that both CD4(+) and CD8(+) cells are crucial for therapy. Lastly, we have established cell clones of other solid tumour cell lines (RM-1, LLC1 and moto1.1) that produce IL-12. Our results show that the delivery of IL-12 by cancer cells is an effective route for immune activation. PMID:24251770

  16. Localized interleukin-12 delivery for immunotherapy of solid tumours

    PubMed Central

    Wei, Louis Z; Xu, Yixin; E Nelles, Megan; Furlonger, Caren; Wang, James CM; Di Grappa, Marco A; Khokha, Rama; Medin, Jeffrey A; Paige, Christopher J

    2013-01-01

    Interleukin (IL)-12 is the key cytokine in the initiation of a Th1 response and has shown promise as an anti-cancer agent; however, clinical trials involving IL-12 have been unsuccessful due to toxic side-effects. To address this issue, lentiviral vectors were used to transduce tumour cell lines that were injected as an autologous tumour cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumour model. SCCVII cells that were transduced to produce IL-12 at different concentrations were then isolated. Subcutaneous injection of parental SCCVII cells results in tumour development, while a mixture of IL-12-producing and non-producing cells results in tumour clearance. Interestingly, when comparing mice injected a mixture of SCCVII and either high IL-12-producing tumour cells or low IL-12-producing tumour cells, we observed that mixtures containing small amounts of high producing cells lead to tumour clearance, whereas mixtures containing large amounts of low producing cells fail to elicit protection, despite the production of equal amounts of total IL-12 in both mixtures. Furthermore, immunizing mice with IL-12-producing cells leads to the establishment of both local and systemic immunity against challenge with SCCVII. Using depletion antibodies, it was shown that both CD4+ and CD8+ cells are crucial for therapy. Lastly, we have established cell clones of other solid tumour cell lines (RM-1, LLC1 and moto1.1) that produce IL-12. Our results show that the delivery of IL-12 by cancer cells is an effective route for immune activation. PMID:24251770

  17. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults

    PubMed Central

    Ingabire, Rosine; Nanvubya, Annet; Anzala, Omu; Karita, Etienne; Hayes, Peter; Kopycinski, Jakub; Dally, Len; Hannaman, Drew; Egan, Michael A.; Eldridge, John H.; Syvertsen, Kristen; Lehrman, Jennifer; Rasmussen, Beth; Gilmour, Jill; Cox, Josephine H.; Fast, Patricia E.; Schmidt, Claudia

    2015-01-01

    Background Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study. Methods Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM. Results All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime—Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected. Conclusion The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected. Trial Registration ClinicalTrials.gov NCT01496989 PMID:26252526

  18. The movement protein of barley yellow dwarf virus-GAV self-interacts and forms homodimers in vitro and in vivo.

    PubMed

    Xia, Zongliang; Cao, Rufei; Sun, Kaile; Zhang, Hua

    2012-07-01

    The 17-kDa movement protein (MP) of the GAV strain of barley yellow dwarf virus (BYDV-GAV) can bind the viral RNA and target to the nucleus. However, much less is known about the active form of the MP in planta. In this study, the ability of the MP to self-interact was analyzed by yeast two-hybrid assay and bimolecular fluorescence complementation. The BYDV-GAV MP has a strong potential to self-interact in vitro and in vivo, and self-interaction was mediated by the N-terminal domain spanning the second α-helix (residues 17-39). Chemical cross-linking and heterologous MP expression from a pea early browning virus (PEBV) vector further showed that MP self-interacts to form homodimers in vitro and in planta. Interestingly, the N-terminal domain necessary for MP self-interaction has previously been identified as important for nuclear targeting. Based on these findings, a functional link between MP self-interaction and nuclear targeting is discussed. PMID:22437255

  19. Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer.

    PubMed Central

    Rasooly, A; Wang, P Z; Novick, R P

    1994-01-01

    The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer. Images PMID:7957090

  20. Treatment with a Monoclonal Anti-IL-12p40 Antibody Induces Substantial Gut Microbiota Changes in an Experimental Colitis Model.

    PubMed

    Castro-Mejía, Josué; Jakesevic, Maja; Krych, Łukasz; Nielsen, Dennis S; Hansen, Lars H; Sondergaard, Bodil C; Kvist, Peter H; Hansen, Axel K; Holm, Thomas L

    2016-01-01

    Background and Aim. Crohn's disease is associated with gut microbiota (GM) dysbiosis. Treatment with the anti-IL-12p40 monoclonal antibody (12p40-mAb) has therapeutic effect in Crohn's disease patients. This study addresses whether a 12p40-mAb treatment influences gut microbiota (GM) composition in mice with adoptive transfer colitis (AdTr-colitis). Methods. AdTr-colitis mice were treated with 12p40-mAb or rat-IgG2a or NaCl from days 21 to 47. Disease was monitored by changes in body weight, stool, endoscopic and histopathology scores, immunohistochemistry, and colonic cytokine/chemokine profiles. GM was characterized through DGGE and 16S rRNA gene-amplicon high-throughput sequencing. Results. Following 12p40-mAb treatment, most clinical and pathological parameters associated with colitis were either reduced or absent. GM was shifted towards a higher Firmicutes-to-Bacteroidetes ratio compared to rat-IgG2a treated mice. Significant correlations between 17 bacterial genera and biological markers were found. The relative abundances of the RF32 order (Alphaproteobacteria) and Akkermansia muciniphila were positively correlated with damaged histopathology and colonic inflammation. Conclusions. Shifts in GM distribution were observed with clinical response to 12p40-mAb treatment, whereas specific GM members correlated with colitis symptoms. Our study implicates that specific changes in GM may be connected with positive clinical outcomes and suggests preventing or correcting GM dysbiosis as a treatment goal in inflammatory bowel disease. PMID:26880890

  1. Treatment with a Monoclonal Anti-IL-12p40 Antibody Induces Substantial Gut Microbiota Changes in an Experimental Colitis Model

    PubMed Central

    Castro-Mejía, Josué; Jakesevic, Maja; Krych, Łukasz; Nielsen, Dennis S.; Hansen, Lars H.; Sondergaard, Bodil C.; Kvist, Peter H.; Hansen, Axel K.; Holm, Thomas L.

    2016-01-01

    Background and Aim. Crohn's disease is associated with gut microbiota (GM) dysbiosis. Treatment with the anti-IL-12p40 monoclonal antibody (12p40-mAb) has therapeutic effect in Crohn's disease patients. This study addresses whether a 12p40-mAb treatment influences gut microbiota (GM) composition in mice with adoptive transfer colitis (AdTr-colitis). Methods. AdTr-colitis mice were treated with 12p40-mAb or rat-IgG2a or NaCl from days 21 to 47. Disease was monitored by changes in body weight, stool, endoscopic and histopathology scores, immunohistochemistry, and colonic cytokine/chemokine profiles. GM was characterized through DGGE and 16S rRNA gene-amplicon high-throughput sequencing. Results. Following 12p40-mAb treatment, most clinical and pathological parameters associated with colitis were either reduced or absent. GM was shifted towards a higher Firmicutes-to-Bacteroidetes ratio compared to rat-IgG2a treated mice. Significant correlations between 17 bacterial genera and biological markers were found. The relative abundances of the RF32 order (Alphaproteobacteria) and Akkermansia muciniphila were positively correlated with damaged histopathology and colonic inflammation. Conclusions. Shifts in GM distribution were observed with clinical response to 12p40-mAb treatment, whereas specific GM members correlated with colitis symptoms. Our study implicates that specific changes in GM may be connected with positive clinical outcomes and suggests preventing or correcting GM dysbiosis as a treatment goal in inflammatory bowel disease. PMID:26880890

  2. An Interleukin-6 Receptor-dependent Molecular Switch Mediates Signal Transduction of the IL-27 Cytokine Subunit p28 (IL-30) via a gp130 Protein Receptor Homodimer*

    PubMed Central

    Garbers, Christoph; Spudy, Björn; Aparicio-Siegmund, Samadhi; Waetzig, Georg H.; Sommer, Jan; Hölscher, Christoph; Rose-John, Stefan; Grötzinger, Joachim; Lorenzen, Inken; Scheller, Jürgen

    2013-01-01

    IL-27 consists of the cytokine subunit p28 and the non-signaling α-receptor EBI3. p28 was shown to additionally act via the non-signaling membrane-bound IL-6 α-receptor (IL-6R) as an agonistic cytokine but also as a gp130 β-receptor antagonist, leading to inhibition of IL-6 signaling. Here, we developed a strategy for bacterial expression, purification, and refolding of murine p28. We show that p28 did not interfere with IL-6- or IL-27-induced signaling, indicating that p28 has no antagonistic properties. Moreover, we demonstrate that murine p28 acts as an agonistic cytokine via the murine and human IL-6R, indicating that p28 exhibits no species specificity. p28 was able to induce p28-trans-signaling via the soluble IL-6R (sIL-6R), a characteristic property that was initially described for trans-signaling of IL-6 via the sIL-6R. Of notice, p28/sIL-6R trans-signaling was inhibited by the IL-6 trans-signaling antagonist, soluble gp130. At higher concentrations, p28 but not IL-6 was able to induce signaling even in the absence of IL-6R or EBI3. Although IL-27 signals via a heterodimer of the β-receptor chains gp130 and Wsx-1, p28/IL-6R specifically recruits two gp130 β-receptor chains for signal transduction. The binding of p28 to a gp130/Wsx-1 heterodimer or a gp130 homodimer is highly selective and controlled by a novel molecular switch induced by EBI3 or IL-6R, respectively. PMID:23209286

  3. IL-10-induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.

    PubMed

    Rossato, Marzia; Curtale, Graziella; Tamassia, Nicola; Castellucci, Monica; Mori, Laura; Gasperini, Sara; Mariotti, Barbara; De Luca, Mariacristina; Mirolo, Massimiliano; Cassatella, Marco A; Locati, Massimo; Bazzoni, Flavia

    2012-11-01

    IL-10 is a potent anti-inflammatory molecule that, in phagocytes, negatively targets cytokine expression at transcriptional and posttranscriptional levels. Posttranscriptional checkpoints also represent the specific target of a recently discovered, evolutionary conserved class of small silencing RNAs known as "microRNAs" (miRNAs), which display the peculiar function of negatively regulating mRNA processing, stability, and translation. In this study, we report that activation of primary human monocytes up-regulates the expression of miR-187 both in vitro and in vivo. Accordingly, we identify miR-187 as an IL-10-dependent miRNA playing a role in IL-10-mediated suppression of TNF-α, IL-6, and the p40 subunit of IL-12 (IL-12p40) produced by primary human monocytes following activation of Toll-like receptor 4 (TLR4). Ectopic expression of miR-187 consistently and selectively reduces TNFα, IL-6, and IL-12p40 produced by LPS-activated monocytes. Conversely, the production of LPS-induced TNF-α, IL-6, and IL-12p40 is increased significantly when miR-187 expression is silenced. Our data demonstrate that miR-187 directly targets TNF-α mRNA stability and translation and indirectly decreases IL-6 and IL-12p40 expression via down-modulation of IκBζ, a master regulator of the transcription of these latter two cytokines. These results uncover an miRNA-mediated pathway controlling cytokine expression and demonstrate a central role of miR-187 in the physiological regulation of IL-10-driven anti-inflammatory responses. PMID:23071313

  4. 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

  5. The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop

    PubMed Central

    Sasseville, Louis J.; Morin, Michael; Coady, Michael J.; Blunck, Rikard; Lapointe, Jean-Yves

    2016-01-01

    Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence “quencher”) to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12–13 loop is located on the external side of the membrane. As the 12–13 loop begins on the intracellular side of the membrane, this suggests that the 12–13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12–13 loop is re-entrant and readily accessible from the extracellular milieu. PMID:27137918

  6. The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop.

    PubMed

    Sasseville, Louis J; Morin, Michael; Coady, Michael J; Blunck, Rikard; Lapointe, Jean-Yves

    2016-01-01

    Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence "quencher") to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12-13 loop is located on the external side of the membrane. As the 12-13 loop begins on the intracellular side of the membrane, this suggests that the 12-13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12-13 loop is re-entrant and readily accessible from the extracellular milieu. PMID:27137918

  7. High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.

    PubMed Central

    Rye, H S; Quesada, M A; Peck, K; Mathies, R A; Glazer, A N

    1991-01-01

    Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes. Images PMID:2014172

  8. The role of charge and multiple faces of the CD8 alpha/alpha homodimer in binding to major histocompatibility complex class I molecules: support for a bivalent model.

    PubMed Central

    Giblin, P A; Leahy, D J; Mennone, J; Kavathas, P B

    1994-01-01

    The CD8 dimer interacts with the alpha 3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8 alpha/alpha homodimer that are likely to be involved in binding to class I. Several key residues are situated on the top face of the dimer within loops analogous to the complementarity-determining regions (CDRs) of immunoglobulin. In addition, other important amino acids are located in the A and B beta-strands on the sides of the dimer. The potential involvement of amino acids on both the top and the side faces of the molecule is consistent with a bivalent model for the interaction between a single CD8 alpha/alpha homodimer and two class I molecules and may have important implications for signal transduction in class I-expressing cells. This study also demonstrates a role for the positive surface potential of CD8 in class I binding and complements previous work demonstrating the importance of a negatively charged loop on the alpha 3 domain of class I for CD8 alpha/alpha-class I interaction. We propose a model whereby residues located on the CDR-like loops of the CD8 homodimer interact with the alpha 3 domain of MHC class I while amino acids on the side of the molecule containing the A and B beta-strands contact the alpha 2 domain of class I. Images PMID:8127870

  9. Novel T-cell epitopes on Schistosoma japonicum SjP40 protein and their preventive effect on allergic asthma in mice.

    PubMed

    Ren, Jiling; Hu, Lizhi; Yang, Jing; Yang, Liang; Gao, Fei; Lu, Ping; Fan, Mengyu; Zhu, Yunjuan; Liu, Junyan; Chen, Lingling; Gupta, Shimpy; Yang, Xi; Liu, Peimei

    2016-05-01

    Allergic asthma is a chronic inflammatory disease mediated by Th2 cell immune responses. Currently, immunotherapies based on immune deviation are attractive, preventive, and therapeutic strategies for asthma. Many studies have shown that intracellular bacterial infections such as mycobacteria and their components can suppress asthmatic reactions by enhancing Th1 responses, while helminth infections and their proteins can inhibit allergic asthma via immune regulation. However, some helminth proteins such as SmP40, the major egg antigen of Schistosoma mansoni, are found as Th1 type antigens. Using a panel of overlapping peptides, we identified T-cell epitopes on SjP40 protein of Schistosoma japonicum, which can induce Th1 cytokine and inhibit the production of Th2 cytokines and airway inflammation in a mouse model of allergic asthma. These results reveal a novel form of immune protective mechanism, which may play an important role in the modulating effect of helminth infection on allergic asthmatic reactions. PMID:26840774

  10. p40 as a Basal Cell Marker in the Diagnosis of Prostate Glandular Proliferations: A Comparative Immunohistochemical Study with 34betaE12

    PubMed Central

    Brustmann, Hermann

    2015-01-01

    Immunohistochemistry is important for the accurate diagnosis of basal cells in atypical glandular proliferations of the prostate. p40, an isoform of p63, may be an adjunct to a marker panel in this setting. Biopsies of 68 patients were analyzed by immunohistochemistry using antibodies to 34betaE12 and p40. Basal cell staining was classified as negative, partial (<60%), or diffuse (≥60%); irregular staining was defined as discordant staining patterns. In acinar proliferations (N = 41), partial staining for both markers was seen in 42%, and diffuse staining in 46% of reactive cases. An irregular reactivity was noted in one case only (2%). Finally, these lesions were signed out as benign. Acinar proliferations negative for both markers and limited amount of glands (≤4) were termed atypical small acinar proliferations (ASAP). Out of six PIN lesions two cases showed partial, three cases showed diffuse reactivity for both markers, and one case was stained irregular. All cases diagnosed as prostate carcinomas (N = 20) had no evidence of basal cell staining for neither of the markers. p40 expression is closely correlated to 34betaE12 with respect to demonstration of basal cells of prostate glands and may provide further information on the dignity of glandular proliferations of the prostate. PMID:25852959

  11. Two markers, IS901-IS902 and p40, identified by PCR and by using monoclonal antibodies in Mycobacterium avium strains.

    PubMed Central

    Ahrens, P; Giese, S B; Klausen, J; Inglis, N F

    1995-01-01

    The occurrence of two markers, a newly identified 40-kDa protein (p40) and the insertion sequence IS901-IS902, in strains of Mycobacterium avium subspp. was evaluated. Analysis of 184 type and field strains of the M. avium complex from human, animal, and environmental sources by PCR specific to IS901 and by a monoclonal antibody specific to p40 demonstrated the presence of the two molecular markers in all of the M. avium subsp. silvaticum strains examined and also in a number of M. avium subsp. avium strains (the latter isolated mainly from pigs). The appearance of the two markers was completely concurrent in all strains. Further, the marker-positive M. avium subsp. avium strains were mainly serotype 2, whereas M. avium complex strains of serotypes 4, 6, 8, 9, and 10 were marker negative. The M. avium subsp. avium type strains ATCC 25291 and approximately 50% of the M. avium subsp. avium field strains isolated from animals contained the markers, while only one strain of human origin was found to be marker positive. Therefore, IS901 and p40 appear to have substantial potential to differentiate among isolates of the M. avium complex. This observation raises new issues regarding classification of strains, since the presence of the markers was found to be inconsistent with the present taxonomic grouping of M. avium subspp. PMID:7615703

  12. Prostaglandin E2 inhibits IL-23 and IL-12 production by human monocytes through down-regulation of their common p40 subunit.

    PubMed

    Kalim, Khalid W; Groettrup, Marcus

    2013-03-01

    The heterodimeric cytokine IL-23 is important for the maintenance of Th17 cells, which are pivotal mediators of autoimmune diseases like rheumatoid arthritis, colitis, and multiple sclerosis. Prostaglandin E2 (PGE2) is a soluble regulator of inflammation that has both pro- and anti-inflammatory properties. PGE2 has been shown to elevate the IL-23 production by dendritic cells (DC). Monocytes are also producers of IL-23 but the effect of PGE2 on IL-23 production by human monocytes has hardly been investigated. We show here that PGE2 blocks the production of IL-23 by LPS-stimulated monocytes in an IL-10 and IL-1β independent manner. This effect was due to the down-regulation of the p40 subunit of IL-23 on mRNA and protein level. The p40 subunit is shared by IL-12 and, consistently, PGE2 also lowered the IL-12 production by monocytes. These effects of PGE2 were cAMP-dependent since the cAMP enhancer forskolin strongly reduced IL-23 and IL-12 production by monocytes. Taken together, PGE2 acts in an anti-inflammatory manner by lowering IL-23 production by monocytes while it has the opposite effect in DC. Our data may help to reconcile controversial point of views on the pro- and anti-inflammatory nature of PGE2 by making a strong case for a cell type-dependent function. PMID:22982753

  13. 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. PMID:27183598

  14. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    PubMed Central

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  15. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway.

    PubMed

    Calegari-Silva, Teresa C; Vivarini, Áislan C; Miqueline, Marina; Dos Santos, Guilherme R R M; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G

    2015-09-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  16. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-01

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis ( YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to α/β proteins, and its topology is a three-layer α/β/α sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% β strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium ( StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli ( EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  17. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    SciTech Connect

    Lashkov, A. A. Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-15

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis (YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to {alpha}/{beta} proteins, and its topology is a three-layer {alpha}/{beta}/{alpha} sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% {beta} strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium (StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli (EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  18. IL-12p40/IL-10 Producing preCD8α/Clec9A+ Dendritic Cells Are Induced in Neonates upon Listeria monocytogenes Infection

    PubMed Central

    Delbauve, Sandrine; Caminschi, Irina; Lahoud, Mireille H.; Shortman, Ken; Flamand, Véronique

    2016-01-01

    Infection by Listeria monocytogenes (Lm) causes serious sepsis and meningitis leading to mortality in neonates. This work explored the ability of CD11chigh lineage DCs to induce CD8+ T-cell immune protection against Lm in mice before 7 days of life, a period symbolized by the absence of murine IL-12p70-producing CD11chighCD8α+ dendritic cells (DCs). We characterized a dominant functional Batf3-dependent precursor of CD11chigh DCs that is Clec9A+CD205+CD24+ but CD8α- at 3 days of life. After Lm-OVA infection, these pre-DCs that cross-present Ag display the unique ability to produce high levels of IL-12p40 (not IL-12p70 nor IL-23), which enhances OVA-specific CD8+ T cell response, and regulatory IL-10 that limits OVA-specific CD8+ T cell response. Targeting these neonatal pre-DCs for the first time with a single treatment of anti-Clec9A-OVA antibody in combination with a DC activating agent such as poly(I:C) increased the protection against later exposure to the Lm-OVA strain. Poly(I:C) was shown to induce IL-12p40 production, but not IL-10 by neonatal pre-DCs. In conclusion, we identified a new biologically active precursor of Clec9A+ CD8α- DCs, endowed with regulatory properties in early life that represents a valuable target to augment memory responses to vaccines. PMID:27074026

  19. Egg antigen p40 of Schistosoma japonicum promotes senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Xu, Tianhua; Zhu, Dandan; Wang, Jianxin; Huang, Caiqun; Lyu, Lei; Hu, Bin; Sun, Wei; Duan, Yinong

    2016-01-01

    Liver fibrosis is a serious disease that is characterized by the excess deposition of extracellular matrix (ECM) components. Activated hepatic stellate cells (HSCs) are a major source of ECM and serve as a key regulator in liver fibrogenesis. Inactivation of HSCs is essential for liver fibrotic regression. The present study explores the underlying mechanisms of Schistosoma japonicum egg antigen p40 (Sjp40) promoting senescence in HSCs and antifibrosis. For the first time we report that Sjp40 inhibits the activation and proliferation of an immortalized human HSC line (LX-2 cells) and promotes cellular senescence and cell cycle arrest. Sjp40 through action on the STAT3/p53/p21 pathway triggered cellular senescence, while knockdown of p53 or STAT3 partly restored cell senescence. In addition, Sjp40-induced cellular senescence caused LX-2 cells to be more sensitive to a human NK cell line (YT cells). Together these findings provide novel insights into the mechanism of antifibrosis and may have implications for the development of antifibrosis therapies. PMID:27468691

  20. Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4.

    PubMed

    Wang, K S; Frank, D A; Ritz, J

    2000-05-15

    Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-gamma by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rbeta1 and IL-12Rbeta2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2-primed NK cells show enhanced functional responses to IL-12 as measured by IFN-gamma production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer. PMID:10807786

  1. Platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair.

    PubMed Central

    Pierce, G. F.; Tarpley, J. E.; Yanagihara, D.; Mustoe, T. A.; Fox, G. M.; Thomason, A.

    1992-01-01

    Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growth factor beta 1 (rTGF-beta 1), and basic fibroblast growth factor (rbFGF) can accelerate healing of soft tissues. However, little information is available characterizing the components of wound matrix induced by these growth factors and the molecular mechanisms underlying accelerated repair and wound maturation. In this study, the composition, quantity, and rate of extracellular matrix deposition within growth factor-treated lapine ear excisional wounds were analyzed at different stages of healing using specific histochemical and immunohistochemical stains, coupled with image analysis techniques. Single application of optimal concentrations of each growth factor accelerated normal healing by 30% (P less than 0.0003); rPDGF-BB markedly augmented early glycosaminoglycan (GAG) and fibronectin deposition, but induced significantly greater levels of collagen later in the repair process, compared with untreated wounds rTGF-beta 1 treatment led to rapidly enhanced collagen synthesis and maturation, without increased GAG deposition. In contrast, rbFGF treatment induced a predominantly angiogenic response in wounds, with a marked increase in endothelia and neovessels (P less than 0.0001), and increased wound collagenolytic activity (P less than 0.03). rbFGF-treated wounds did not evolve into collagen-containing scars and continued to accumulate only provisional matrix well past wound closure. These results provide new evidence that growth factors influence wound repair via different mechanisms: 1) rPDGF-BB accelerates deposition of provisional wound matrix; 2) rTGF-beta 1 accelerates deposition and maturation of collagen; and 3) rbFGF induces a profound monocellular angiogenic response which may lead to a marked delay in wound maturation, and the possible loss of the normal signal(s) required to stop repair. These results suggest that specific growth factors may selectively regulate

  2. Gene therapy approaches against cancer using in vivo and ex vivo gene transfer of interleukin-12.

    PubMed

    Hernandez-Alcoceba, Ruben; Poutou, Joanna; Ballesteros-Briones, María Cristina; Smerdou, Cristian

    2016-02-01

    IL-12 is an immunostimulatory cytokine with strong antitumor properties. Systemic administration of IL-12 in cancer patients led to severe toxic effects, prompting the development of gene therapy vectors able to express this cytokine locally in tumors. Both nonviral and viral vectors have demonstrated a high antitumor efficacy in preclinical tumor models. Some of these vectors, including DNA electroporation, adenovirus and ex vivo transduced dendritic cells, were tested in patients, showing low toxicity and moderate antitumor efficacy. IL-12 activity can be potentiated by molecules with immunostimulatory, antiangiogenic or cytotoxic activity. These combination therapies are of clinical interest because they could lower the threshold for IL-12 efficacy, increasing the therapeutic potential of gene therapy and preventing the toxicity mediated by this cytokine. PMID:26786809

  3. Interleukin-12, Paclitaxel, and Trastuzumab in Treating Patients With Solid Tumors

    ClinicalTrials.gov

    2013-06-03

    Male Breast Cancer; Recurrent Breast Cancer; Recurrent Endometrial Carcinoma; Recurrent Gastric Cancer; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Small Cell Lung Cancer

  4. Interleukin-12 in Treating Patients With Previously Treated Non-Hodgkin's Lymphoma or Hodgkin's Disease

    ClinicalTrials.gov

    2015-04-14

    Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Splenic Marginal Zone Lymphoma; Waldenström Macroglobulinemia

  5. Rituximab and Interleukin-12 in Treating Patients With B-Cell Non-Hodgkin's Lymphoma

    ClinicalTrials.gov

    2013-08-23

    Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Small Lymphocytic Lymphoma; Splenic Marginal Zone Lymphoma

  6. Interleukin-12 in Treating Patients With Hematologic Cancers or Solid Tumors

    ClinicalTrials.gov

    2014-09-09

    Breast Cancer; Chronic Myeloproliferative Disorders; Gestational Trophoblastic Tumor; Kidney Cancer; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Neuroblastoma; Ovarian Cancer; Testicular Germ Cell Tumor

  7. Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity.

    PubMed

    Yang, Lirong; Zaharoff, David A

    2013-05-01

    Local delivery systems that provide sustained, high concentrations of antitumor cytokines in the tumor microenvironment while minimizing systemic dissemination are needed to realize the potential of cytokine-based immunotherapies. Recently, co-formulations of cytokines with chitosan solutions have been shown to increase local cytokine retention and bioactivity. In particular, intratumoral (i.t.) injections of chitosan/IL-12 can eliminate established tumors and generate tumor-specific immune responses. In the present study, we explored the mechanisms by which chitosan potentiated IL-12's antitumor activity. The location of chitosan/IL-12 injection was found to be critical for optimal cytokine delivery. I.t. injections eliminated 9 of 10 MC38 adenocarcinomas while contralateral and peritumoral injections delayed tumor growth but could not eliminate tumors. Microdosing studies demonstrated that IL-12 depots, simulated through daily i.t. injections with IL-12 alone, were not as effective as weekly i.t. chitosan/IL-12. 50-75% of mice receiving daily IL-12 microdoses and 87.5% of mice receiving weekly chitosan/IL-12 were cured of MC38 tumors. Chitosan was found to increase IL-12-mediated leukocytic expansion in tumors and tumor-draining lymph nodes (TDLNs) by 40 and 100%, respectively. Immunophenotyping studies demonstrated that chitosan co-formulation amplified IL-12-induced increases in important effector populations, such as CD8(+)IFN-γ(+) and NKT cells, in tumors and dendritic cell populations in TDLNs. Remarkable increases in Gr-1(+)CD11b(+) tumor infiltrates were also observed in mice receiving chitosan or chitosan/IL-12. This population does not appear be suppressive and may facilitate the local antitumor response. Presented data suggest that chitosan-mediated depot formation and enhanced local cytokine retention is significantly, but not entirely, responsible for increased cytokine bioactivity. PMID:23453060

  8. Interleukin-12 and Interleukin-2 in Treating Patients With Mycosis Fungoides

    ClinicalTrials.gov

    2013-01-15

    Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Stage I Cutaneous T-cell Non-Hodgkin Lymphoma; Stage I Mycosis Fungoides/Sezary Syndrome; Stage II Cutaneous T-cell Non-Hodgkin Lymphoma; Stage II Mycosis Fungoides/Sezary Syndrome; Stage III Cutaneous T-cell Non-Hodgkin Lymphoma; Stage III Mycosis Fungoides/Sezary Syndrome; Stage IV Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IV Mycosis Fungoides/Sezary Syndrome

  9. Interleukin-12 Immunomodulation Delays the Onset of Lethal Peritoneal Disease of Ovarian Cancer.

    PubMed

    Cohen, Courtney A; Shea, Amanda A; Heffron, C Lynn; Schmelz, Eva M; Roberts, Paul C

    2016-01-01

    The omental fat band (OFB) is the predominant site for metastatic seeding of ovarian cancer. Previously, we highlighted the influx and accumulation of neutrophils and macrophages in the OFB following syngeneic ovarian cancer cell seeding as an important factor in the development of a protumorigenic cascade. Here we investigated localized immunomodulation as a means of promoting a successful protective response. As an important TH1-type immunomodulator, interleukin (IL)-12 has previously been investigated clinically as an anticancer therapeutic. However, systemic IL-12 administration was associated with serious side effects, galvanizing the development of immune or accessory cells engineered to express secreted or membrane-bound IL-12 (mbIL-12). Using an mbIL-12-expressing cell variant, we demonstrate that localized IL-12 in the tumor microenvironment significantly delays disease development. The mbIL-12-mediated decrease in tumor burden was associated with a significant reduction in neutrophil and macrophage infiltration in the OFB, and correlated with a reduced expression of neutrophil and macrophage chemoattractants (CXCL1, -2, -3 and CCL2, -7). Vaccination with mitotically impaired tumor cells did not confer protection against subsequent tumor challenge, indicating that IL-12 did not impact the immunogenicity of the cancer cells. Our findings are in agreement with previous reports suggesting that IL-12 may hold promise when delivered in a targeted and sustained manner to the omental microenvironment. Furthermore, resident cells within the omental microenvironment may provide a reservoir that can be activated and mobilized to prevent metastatic seeding within the peritoneum and, therefore, may be targets for chemotherapeutics. PMID:26430781

  10. LOWER LEVELS OF INTERLEUKIN-12 PRECEDE THE DEVELOPMENT OF TUBERCULOSIS AMONG HIV-INFECTED WOMEN

    PubMed Central

    Bordón, José; Plankey, Michael W.; Young, Mary; Greenblatt, Ruth M.; Villacres, Maria C.; French, Audrey L.; Zhang, Jie; Brock, Guy; Appana, Savitri; Herold, Betsy; Durkin, Helen; Golub, Jonathan E.; Fernandez-Botran, Rafael

    2012-01-01

    Tuberculosis (TB) is the worldwide leading cause of death among HIV-infected individuals, accounting for more than half of AIDS-related deaths. A high risk of tuberculosis (TB) has been shown in early stages of the HIV disease, even in the presence of normal CD4+ cell counts. Moreover, the factors that determine protective immunity vs. susceptibility to M. tuberculosis cannot be fully explained by simple changes in IFNγ levels or a shift from Th1 to Th2 cytokines. This work investigated the relationship between cytokine expression profiles in peripheral blood mononuclear cells (PBMC) and susceptibility to M. tuberculosis in ten HIV+ women who went on to develop TB. RNA transcripts for IL-4, IL-4δ2, IL-10, IL-12(p35), IL-13, IL-17A, IFNγ and TNFα were measured by real-time quantitative PCR in unstimulated or TB peptide antigen-stimulated PBMCs from ten HIV+ women with positive tuberculin skin tests (TST) and compared with HIV-seropositive and seronegative women without previous TB and negative TST. Stimulated PBMC cultures showed significantly lower expression of IL-12p35 (p=0.004) and IL-10 (p=0.026) in the HIV+TB+ group six to twelve months before onset of TB compared to HIV+TB− women. Unstimulated PBMC from HIV+TB+ women also had lower expression of Th2 cytokines [IL-4 (p=0.056) and IL-13 (p=0.050)] compared to HIV+TB− women. These results suggest that lower IL-12 production by PBMC in response to TB antigens and lower levels of both Th1 and Th2 cytokines by PBMC correlate with future development of TB in HIV-infected women and may be responsible for their increased susceptibility. PMID:21880503

  11. Interleukin-12 Followed by Interferon Alfa in Treating Patients With Advanced Cancer

    ClinicalTrials.gov

    2013-01-31

    Chronic Myeloproliferative Disorders; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Precancerous Condition; Unspecified Adult Solid Tumor, Protocol Specific

  12. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

    PubMed Central

    Caruso, M; Pham-Nguyen, K; Kwong, Y L; Xu, B; Kosai, K I; Finegold, M; Woo, S L; Chen, S H

    1996-01-01

    Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma. Images Fig. 5 PMID:8876130

  13. Utility of a novel triple marker (combination of thyroid transcription factor 1, Napsin A, and P40) in the subclassification of non-small cell lung carcinomas using fine-needle aspiration cases.

    PubMed

    Sharma, Rajni; Wang, Yuting; Chen, Li; Gurda, Grzegorz T; Geddes, Susan; Gabrielson, Edward; Askin, Frederic; Li, Qing Kay

    2016-08-01

    Personalized treatment of lung cancer requires an accurate subclassification of non-small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC), squamous cell carcinoma (SqCC), and other subtypes. In poorly differentiated tumors especially on small fine-needle aspirate specimens, the subclassification could be difficult in certain cases. Our previous study using resected tumor tissue has shown that the combination of commonly used individual markers (thyroid transcription factor 1 [TTF-1], P40, and Napsin A) into a novel triple marker has high sensitivity and specificity in subclassification of NSCLC and also the advantage of using minimal tumor tissue. In this study, we further evaluated the utility of this novel triple marker using fine-needle aspirate cases. We included primary NSCLC, consisting of 37 SqCCs (primary, 35; metastasis, 2) and 50 ADCs (primary, 29; metastasis, 21), 12 metastatic ADCs of nonpulmonary primary, and 10 small cell lung carcinomas. The immunohistochemical patterns were semiquantitatively scored. In lung SqCCs and ADCs, the sensitivity and specificity of the triple marker were 100% and 97.1% and 86.0% and 100%, respectively. The triple marker showed no immunoreactivity in 12 metastatic nonpulmonary ADCs. In 10 small cell lung carcinomas, TTF-1 had focal positivity in 40% of cases. The limitations of the triple marker include staining of alveolar macrophages (by TTF-1 and Napsin A), basal layer of bronchial epithelial cells (by P40), and nonspecific cytoplasmic staining of TTF-1. Our study not only supports our previous finding using resected tumor specimens but also provides evidence that the triple marker can be used for cytological material and preserving tumor tissue for molecular testing. PMID:27045515

  14. 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 Central

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

    2013-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions and Significance 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

  15. Interleukin-12 is synthesized by mesangial cells and stimulates platelet-activating factor synthesis, cytoskeletal reorganization, and cell shape change.

    PubMed

    Bussolati, B; Mariano, F; Biancone, L; Foà, R; David, S; Cambi, V; Camussi, G

    1999-02-01

    Preliminary studies indicate the involvement of interleukin (IL)-12 in experimental renal pathology. In the present study, we evaluated whether cultured glomerular mesangial cells are able to produce IL-12 and whether IL-12 may regulate some of their functions, including the cytoskeletal reorganization, the change in cell shape, and the production of platelet-activating factor (PAF). The results obtained indicate that pro-inflammatory stimuli, such as tumor necrosis factor-alpha and bacterial polysaccharides, induce the expression of IL-12 mRNA and the synthesis of the protein by cultured mesangial cells. Moreover, cultured mesangial cells were shown to bind IL-12 and to express the human low-affinity IL-12 beta1-chain receptor. When challenged with IL-12, mesangial cells produced PAF in a dose- and time-dependent manner and superoxide anions. No production of tumor necrosis factor-alpha and IL-8 was observed. Moreover, we demonstrate that IL-12 induced a delayed and sustained shape change of mesangial cells that reached its maximum between 90 and 120 minutes of incubation. The changes in cell shape occurred concomitantly with cytoskeletal rearrangements and may be consistent with cell contraction. As IL-12-dependent shape change of mesangial cells was concomitant with the synthesis of PAF, which is known to promote mesangial cell contraction, we investigated the role of PAF using two chemically different PAF receptor antagonists. Both antagonists inhibited almost completely the cell shape change induced by IL-12, whereas they were ineffective on angiotensin-II-induced cell shape change. In conclusion, our results suggest that mesangial cells can either produce IL-12 or be stimulated by this cytokine to synthesize PAF and to undergo shape changes compatible with cell contraction. PMID:10027419

  16. Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

    PubMed

    Su, Bor Sheu; Chiu, Hua Hsien; Lin, Cheng Chung; Shien, Jui Hung; Yin, Hsien Sheng; Lee, Long Huw

    2011-02-15

    A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen. PMID:21035196

  17. Interleukin-12 and Trastuzumab in Treating Patients With Cancer That Has High Levels of HER2/Neu

    ClinicalTrials.gov

    2013-02-27

    Advanced Adult Primary Liver Cancer; Anaplastic Thyroid Cancer; Bone Metastases; Carcinoma of the Appendix; Distal Urethral Cancer; Fallopian Tube Cancer; Gastrinoma; Glucagonoma; Inflammatory Breast Cancer; Insulinoma; Liver Metastases; Localized Unresectable Adult Primary Liver Cancer; Lung Metastases; Male Breast Cancer; Malignant Pericardial Effusion; Malignant Pleural Effusion; Metastatic Gastrointestinal Carcinoid Tumor; Metastatic Parathyroid Cancer; Metastatic Transitional Cell Cancer of the Renal Pelvis and Ureter; Newly Diagnosed Carcinoma of Unknown Primary; Occult Non-small Cell Lung Cancer; Pancreatic Polypeptide Tumor; Primary Peritoneal Cavity Cancer; Proximal Urethral Cancer; Pulmonary Carcinoid Tumor; Recurrent Adenoid Cystic Carcinoma of the Oral Cavity; Recurrent Adrenocortical Carcinoma; Recurrent Adult Primary Liver Cancer; Recurrent Anal Cancer; Recurrent Bladder Cancer; Recurrent Breast Cancer; Recurrent Carcinoma of Unknown Primary; Recurrent Cervical Cancer; Recurrent Colon Cancer; Recurrent Endometrial Carcinoma; Recurrent Esophageal Cancer; Recurrent Extrahepatic Bile Duct Cancer; Recurrent Gallbladder Cancer; Recurrent Gastric Cancer; Recurrent Gastrointestinal Carcinoid Tumor; Recurrent Islet Cell Carcinoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Mucoepidermoid Carcinoma of the Oral Cavity; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Pancreatic Cancer; Recurrent Parathyroid Cancer; Recurrent Prostate Cancer; Recurrent Rectal Cancer; Recurrent Renal Cell Cancer; Recurrent Salivary Gland Cancer; Recurrent Small Intestine Cancer; Recurrent Squamous Cell Carcinoma of the Larynx; Recurrent Squamous Cell Carcinoma of the Lip and Oral Cavity; Recurrent Squamous Cell Carcinoma of the Nasopharynx; Recurrent Squamous Cell Carcinoma of the Oropharynx; Recurrent Thyroid Cancer; Recurrent Transitional Cell Cancer of the Renal Pelvis and Ureter; Recurrent Urethral Cancer; Recurrent Vaginal Cancer; Recurrent Vulvar Cancer; Skin Metastases; Small Intestine Adenocarcinoma; Somatostatinoma; Stage III Adenoid Cystic Carcinoma of the Oral Cavity; Stage III Adrenocortical Carcinoma; Stage III Bladder Cancer; Stage III Cervical Cancer; Stage III Colon Cancer; Stage III Endometrial Carcinoma; Stage III Esophageal Cancer; Stage III Follicular Thyroid Cancer; Stage III Gastric Cancer; Stage III Malignant Testicular Germ Cell Tumor; Stage III Mucoepidermoid Carcinoma of the Oral Cavity; Stage III Ovarian Epithelial Cancer; Stage III Pancreatic Cancer; Stage III Papillary Thyroid Cancer; Stage III Prostate Cancer; Stage III Rectal Cancer; Stage III Renal Cell Cancer; Stage III Salivary Gland Cancer; Stage III Squamous Cell Carcinoma of the Larynx; Stage III Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage III Squamous Cell Carcinoma of the Nasopharynx; Stage III Squamous Cell Carcinoma of the Oropharynx; Stage III Vaginal Cancer; Stage III Vulvar Cancer; Stage IIIA Anal Cancer; Stage IIIA Breast Cancer; Stage IIIA Non-small Cell Lung Cancer; Stage IIIB Anal Cancer; Stage IIIB Breast Cancer; Stage IIIB Non-small Cell Lung Cancer; Stage IV Adenoid Cystic Carcinoma of the Oral Cavity; Stage IV Adrenocortical Carcinoma; Stage IV Anal Cancer; Stage IV Bladder Cancer; Stage IV Breast Cancer; Stage IV Colon Cancer; Stage IV Endometrial Carcinoma; Stage IV Esophageal Cancer; Stage IV Follicular Thyroid Cancer; Stage IV Gastric Cancer; Stage IV Mucoepidermoid Carcinoma of the Oral Cavity; Stage IV Non-small Cell Lung Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Pancreatic Cancer; Stage IV Papillary Thyroid Cancer; Stage IV Prostate Cancer; Stage IV Rectal Cancer; Stage IV Renal Cell Cancer; Stage IV Salivary Gland Cancer; Stage IV Squamous Cell Carcinoma of the Larynx; Stage IV Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage IV Squamous Cell Carcinoma of the Nasopharynx; Stage IV Squamous Cell Carcinoma of the Oropharynx; Stage IVA Cervical Cancer; Stage IVA Vaginal Cancer; Stage IVB Cervical Cancer; Stage IVB Vaginal Cancer; Stage IVB Vulvar

  18. Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases.

    PubMed

    Floss, Doreen M; Klöcker, Tobias; Schröder, Jutta; Lamertz, Larissa; Mrotzek, Simone; Strobl, Birgit; Hermanns, Heike; Scheller, Jürgen

    2016-07-15

    The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable. PMID:27193299

  19. Interleukin-18, interleukin-12B and interferon-γ gene polymorphisms in Brazilian patients with rheumatoid arthritis: a pilot study.

    PubMed

    Angelo, H D; Gomes Silva, I I F; Oliveira, R D R; Louzada-Júnior, P; Donadi, E A; Crovella, S; Maia, M M D; de Souza, P R E; Sandrin-Garcia, P

    2015-10-01

    Polymorphisms in interleukin (IL)-18, IL-12 and interferon (IFN)-γ genes are associated with different levels of cytokines expression and have been associated with rheumatoid arthritis (RA). IL-18 +105 A/C, IL-12B +1188 A/C and IFN-γ +874 T/A polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction (PCR) and amplification refractory mutation system PCR from 90 RA patients and 186 healthy individuals. There were significant differences to IL-18 +105 A/C polymorphism between the RA and control groups (odds ratio = 3.77; P < 0.0001). Individual carriers of the variant allele C had a 3.77-fold increased risk of for RA (P = 0.0032). No association was observed for IL-12B and IFN-γ polymorphisms. Our finds suggest a possible role for IL-18 polymorphism in the RA susceptibility in studied population. PMID:26302971

  20. Modulation of host responses to blood-stage malaria by interleukin-12: from therapy to adjuvant activity.

    PubMed

    Stevenson, M M; Su, Z; Sam, H; Mohan, K

    2001-01-01

    This review focuses on the role of interleukin (IL)-12, a proinflammatory cytokine with pleiotropic effects as a potent immunoregulatory molecule and hematopoietic growth factor, in infection with Plasmodium parasites, the causative agents of malaria. IL-12 has been demonstrated to have profound effects on the immune response to blood-stage malaria, to induce protection, and to alleviate malarial anemia. In combination with an anti-malarial drug, IL-12 is effective in an established malaria infection. This cytokine also has potent immune effects as a malaria vaccine adjuvant. However, IL-12 can also mediate pathology during blood-stage malaria. PMID:11226854

  1. A derivative of Lactococcus lactis strain H61 with less interleukin-12 induction has a different cell wall.

    PubMed

    Kimoto-Nira, H; Suzuki, C; Aoki, R; Kobayashi, M; Mizumachi, K

    2012-06-01

    Lactococcus lactis H61 can increase the cellular immune responses of aged (14-mo-old) senescence-accelerated mice. The aim of this study was to investigate the factors contributing to IL-12 induction by strain H61 by analyzing strains derived from it. Strain H61 derivative no. 13 was obtained by growing the parent strain at 37°C. This derivative induced significantly lower production of IL-12 from J774.1 macrophage cells than did the parent strain H61. The 2 strains differed in the resistance of their whole cells or cell walls to lysozyme, a cell wall-degrading enzyme. Sodium hydroxide treatment to de-O-acetylate muramic acid in the cell walls of the 2 strains reduced the lysozyme resistance, compared with untreated cell walls: at 3h after adding lysozyme, the lysozyme resistance of untreated and NaOH treated cell wall from strain H61 was 55.4% and 11.7%, respectively. The values of untreated and NaOH-treated cell walls from strain no.13 were 73.7 and 42.8%, respectively. The reduction was higher in strain H61, indicating that the cell walls of strain H61 were highly O-acetylated. Trichloroacetic acid treatment to remove wall-associated polymers such as teichoic acids made the lysozyme resistance of the cell walls of both strains similar. The sugar content of cell walls prepared from strain H61 was significantly higher than that of strain no. 13 cell wall. A derivative with less activity for inducing IL-12 by macrophage cells had less O-acetylation and had lower sugar content in the cell wall than did strain H61. Modifying the cell wall of strain H61 may be a useful way to regulate its ability to induce IL-12. Strain H61 has been used as a starter bacterium in the dairy industry. This study could lead to enhancing the value of dairy products made by strain H61 by characterizing the key factor(s) responsible for its stimulation of immunity. PMID:22612923

  2. Characterization of a novel subset of CD8(+) T cells that expands in patients receiving interleukin-12.

    PubMed

    Gollob, J A; Schnipper, C P; Orsini, E; Murphy, E; Daley, J F; Lazo, S B; Frank, D A; Neuberg, D; Ritz, J

    1998-08-01

    IL-12 has significant antitumor activity in mice that may be mediated by CD8(+) T cells. We show in this report that repeated subcutaneous injections of IL-12 in patients with cancer resulted in the selective expansion of a subset of peripheral blood CD8(+) T cells. This T cell subset expressed high levels of CD18 and upregulated IL-12 receptor expression after IL-12 treatment in vivo. In normal subjects, these CD3(+)CD8(+)CD18(bright) T cells expressed IL-12 and IL-2 receptors and adhesion/costimulatory molecules to a greater degree than other CD8(+) and CD4(+) T cells. They appeared morphologically as large granular lymphocytes, although they did not express NK cell markers such as CD56. In addition, CD8(+)CD18(bright) T cells were almost exclusively T cell receptor (TCR) alphabeta+, and exhibited a TCR Vbeta repertoire that was strikingly oligoclonal, whereas the Vbeta repertoire of CD18(dim) T cells was polyclonal. Although CD8+CD18(bright) T cells demonstrated little functional responsiveness to IL-12 or IL-2 alone in vitro, they responded to the combination of IL-12+IL-2 with strong IFN-gamma production and proliferation and enhanced non-MHC-restricted cytolytic activity. In contrast, CD18(dim) T cells were not activated by IL-12 or IL-2, alone or in combination. These findings demonstrate that CD8+CD18(bright) T cells are a unique population of peripheral blood lymphocytes with features of both memory and effector cells that are capable of TCR-independent activation through combined stimulation with IL-12+IL-2. As this activation results in IFN-gamma production and enhanced cytolytic activity, these T cells may play a role in innate as well as acquired immunity to tumors and infectious pathogens. Additional studies will be necessary to determine whether CD8+CD18(bright) T cells mediate the antitumor effect of IL-12 or IL-2 administered to cancer patients, and if so, whether maximal activation of these T cells with the combination of IL-12+IL-2 in vivo can augment the clinical effectiveness of these cytokines. PMID:9691093

  3. Parallel topology of genetically fused EmrE homodimers.

    PubMed

    Steiner-Mordoch, Sonia; Soskine, Misha; Solomon, Dalia; Rotem, Dvir; Gold, Ayala; Yechieli, Michal; Adam, Yoav; Schuldiner, Shimon

    2008-01-01

    EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed. PMID:18059473

  4. Non-heat pipe receiver/p-40 Stirling engine

    NASA Technical Reports Server (NTRS)

    Haglund, R. A.

    1981-01-01

    The technology for a full-up hybrid dish-Stirling Solar Thermal Power system is discussed. Overall solar-to-electric efficiency for the dish-Stirling system demonstration is approximately 30%. Hybrid operation is provided by fossil fuel combustion augmentation, which enables the Stirling engine to operate continuously at constant speed and power, regardless of insolation level, thus providing the capability to operate on cloudy days and at night.

  5. Interleukin-12 inhibits the hepatocellular carcinoma growth by inducing macrophage polarization to the M1-like phenotype through downregulation of Stat-3.

    PubMed

    Wang, Qin; Cheng, Feng; Ma, Ting-Ting; Xiong, Hai-Yu; Li, Zi-Wei; Xie, Chang-Li; Liu, Cui-Ying; Tu, Zhi-Guang

    2016-04-01

    Hepatocellular carcinoma is the third most common cause of cancer death worldwide. Novel early detection biomarkers and efficacious therapy strategies are needed. Macrophages recruited from circulation monocytes are the major component of solid cancer and play an important role in the carcinogenesis. Whether overexpression of L-12 in monocytes could induce the phenotype directional differentiation into tumoricidal M1 macrophages and inhibit HCC growth in tumor microenvironment was investigated in this study. For the establishment of the monocyte/IL-12 and polarization of M1-like macrophage, the IL-12 overexpressing recombinant monocyte/IL-12 cells were established by infecting with pAd5F35-CMV/IL-12 adenovirus and co-cultured with HCC SMMC-7721 and Hep3B cells. It was found that the phenotype of monocyte/IL-12 polarized to M1-like macrophages with CD197high IL-12high CD206low IL-10low, and decreased expression of TGF-β, VEGF-A, and MMP-9. In order to explore the mechanism underlying the macrophages polarization, we detected the Stat-3 pathway and its downstream transcription factor c-myc, and found that the p-Stat-3 and c-myc were down-regulated. To evaluate the effects of monocyte/IL-12 on inhibiting HCC growth, various assays including CCK8, flow cytometry, colony-forming and Transwell assays in vitro, and xenograft mouse models and immunohistochemical analyses in vivo were used to detect the HCC growth and relative markers. Treated with IL-12 overexpressing monocytes, the xenograft tumor growth was significantly inhibited in vivo. These results have proven that IL-12-overexpressed monocytes could directionally differentiate to M1-like macrophages through downregulation of Stat-3 and result in the inhibition of HCC growth. PMID:27003285

  6. A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12

    PubMed Central

    1995-01-01

    Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC. PMID:7699334

  7. Innate immune responses in Lyme borreliosis: enhanced tumour necrosis factor-α and interleukin-12 in asymptomatic individuals in response to live spirochetes

    PubMed Central

    Sjöwall, J; Carlsson, A; Vaarala, O; Bergström, S; Ernerudh, J; Forsberg, P; Ekerfelt, C

    2005-01-01

    Innate immunity is important for early defence against borrelia spirochetes and should play a role in the clinical outcome of the infection. In order to study early cytokine responses, in vitro differentiated dendritic cells (DCs) and whole blood cells from 21 patients with different clinical outcomes of Lyme neuroborreliosis were stimulated with live borrelia spirochetes. The borrelia-induced secretion of interleukin (IL)-4, IL-10, IL-12p70, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in DCs and IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β and eotaxin in whole blood cells was measured by enzyme-linked immunospot (ELISPOT) and multiplex arrays, respectively. We found increased numbers of TNF-α-secreting DCs (P = 0·018) in asymptomatic seropositive individuals compared to patients with subacute neuroborreliosis and seronegative controls. Asymptomatic individuals were also found to have elevated levels of IL-12p70 (P = 0·031) in whole blood cell supernatants compared to seronegative controls. These results are in line with previous experiments using cells of the adaptive immune response, indicating that strong T helper type 1 (Th1) proinflammatory responses might be associated with a successful resolution of Lyme disease. PMID:15958074

  8. Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12.

    PubMed

    Wakatsuki, Ayako; Borrow, Persephone; Rigley, Kevin; Beverley, Peter C L

    2003-07-01

    Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response. PMID:12811846

  9. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice.

    PubMed

    Kishida, T; Asada, H; Satoh, E; Tanaka, S; Shinya, M; Hirai, H; Iwai, M; Tahara, H; Imanishi, J; Mazda, O

    2001-08-01

    Direct intratumoral transfection of cytokine genes was performed by means of the in vivo electroporation as a novel therapeutic strategy for cancer. Plasmid vectors carrying the firefly luciferase, interleukin (IL)-12 and IL-18 genes were injected into established subcutaneous B16-derived melanomas followed by electric pulsation. When plasmid vectors with Epstein--Barr virus (EBV) nuclear antigen 1 (EBNA1) gene were employed, the expression levels of the transgenes were significantly higher in comparison with those obtained with conventional plasmid vectors. In consequence of the transfection with IL-12 and IL-18 genes, serum concentrations of the cytokines were significantly elevated, while interferon (IFN)-gamma also increased in the sera of the animals. The IL-12 gene transfection resulted in significant suppression of tumor growth, while the therapeutic effect was further improved by co-transfection with IL-12 and IL-18 genes. Repetitive co-transfection with IL-12 and IL-18 genes resulted in significant prolongation of survival of the animals. Natural killer (NK) and cytotoxic T lymphocyte (CTL) activities were markedly enhanced in the mice transfected with the cytokine genes. The present data suggest that the cytokine gene transfer can be successfully achieved by in vivo electroporation, leading to both specific and nonspecific antitumoral immune responses and significant therapeutic outcome. PMID:11509956

  10. Therapeutic Administration of KM+ Lectin Protects Mice Against Paracoccidioides brasiliensis Infection via Interleukin-12 Production in a Toll-Like Receptor 2-Dependent Mechanism

    PubMed Central

    Coltri, Kely C.; Oliveira, Leandro L.; Pinzan, Camila F.; Vendruscolo, Patrícia E.; Martinez, Roberto; Goldman, Maria Helena; Panunto-Castelo, Ademilson; Roque-Barreira, Maria-Cristina

    2008-01-01

    KM+ is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major infection. In this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM+ (jfKM+) and its recombinant counterpart (rKM+) in experimental paracoccidioidomycosis. To this end, jfKM+ or rKM+ was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM+-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM+-treated mice presented higher levels of nitric oxide, IL-12, interferon-γ, and tumor necrosis factor-α, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM+ led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM+ on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule. PMID:18599609