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Sample records for jnk-mediated interleukin-2 mrna

  1. Substance P stabilizes interleukin-2 mRNA in activated Jurkat cells.

    PubMed

    Calvo, C F

    1994-04-01

    We investigated here the mechanism leading to the enhancement of interleukin (IL)-2 mRNA that we described in a previous work when Jurkat cells were co-stimulated with PHA+PMA and 10(-12) M of the Substance P (SP) neuropeptide. We show that the SP-augmented IL-2 mRNA signal is totally abrogated by an early addition of cyclosporin A, actinomycin D or cycloheximide. SP does not affect the IL-2 gene transcription, as evidenced by nuclear run on assays. In contrast, a posttranscriptional alteration of the IL-2 mRNA is shown, by demonstrating that the degradation rate of IL-2 mRNA following the addition of actinomycin D, at 4 h, was delayed in the (PHA+PMA)-activated cell cultures containing 10(-12) M of SP. Thus, the SP-induced augmentation of secreted IL-2 in activated T cells we demonstrated previously must result from an SP increase of the IL-2 mRNA stability. PMID:7512581

  2. Detection of three nonsense mutations and one missense mutation in the interleukin-2 receptor [gamma] chain gene in SCIDX1 that differently affect the mRNA processing

    SciTech Connect

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de ); Subtil, A.; Dautry-Varsat, A. )

    1994-05-01

    The interleukin-2 receptor [gamma] (IL-2R[gamma]) chain gene encodes a 64-kDa protein that not only composes the high-affinity form of the IL-2 binding receptor in association with the 2R [alpha] and [beta] chains, but also participates in at least the IL-4 and IL-7 receptor complexes. Mutations in this gene have recently been shown to cause X-linked severe combined immunodeficiency (SCIDX1). This disease of the immune system results from an early block of T lymphocyte and natural killer (NK) cell differentiation, which leads to a severe cellular and humoral immune defect that is lethal unless treated by bone marrow transplantation. Analysis of the IL-2R[gamma] gene in SCIDX1 patients has revealed the presence of heterogeneous mutations principally located in the extracellular domain of the molecule. We report here three intraexonic mutations and one deletion in the IL-2R[gamma] gene in four SCIDX1 patients. These mutations appear to differentially affect RNA processing, either by decreasing IL-2R[gamma] mRNA level or by the skipping of a constitutive exon. 16 refs., 1 fig.

  3. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  4. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.

    PubMed Central

    Boumpas, D T; Anastassiou, E D; Older, S A; Tsokos, G C; Nelson, D L; Balow, J E

    1991-01-01

    Glucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production. Images PMID:2022743

  5. A JNK-mediated autophagy pathway that triggers c-IAP degradation and necroptosis for anticancer chemotherapy

    PubMed Central

    He, Weiyang; Wang, Qiong; Srinivasan, Balasubramanian; Xu, Jennings; Padilla, Mabel T.; Li, Zi; Wang, Xia; Liu, Yushi; Gou, Xin; Shen, Han-Ming; Xing, Chengguo; Lin, Yong

    2014-01-01

    Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin1. Importantly, suppression of autophagy, with either pharmacological inhibitors or siRNAs targeting the essential autophagy components ATG7 and Beclin1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance. PMID:23831571

  6. JNK-mediated activation of ATF2 contributes to dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.

    PubMed

    Huang, Qiaoying; Du, Xiaoxiao; He, Xin; Yu, Qing; Hu, Kunhua; Breitwieser, Wolfgang; Shen, Qingyu; Ma, Shanshan; Li, Mingtao

    2016-03-01

    The c-Jun N-terminal kinase (JNK)/c-Jun pathway is a known critical regulator of dopaminergic neuronal death in Parkinson's disease (PD) and is considered a potential target for neuroprotective therapy. However, whether JNK is activated within dopaminergic neurons remains controversial, and whether JNK acts through downstream effectors other than c-Jun to promote dopaminergic neuronal death remains unclear. In this study, we confirm that JNK but not p38 is activated in dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxication. Furthermore, within the dopaminergic neurons of the substantia nigra in MPTP-treated mice, JNK2/3 phosphorylates threonine 69 (Thr69) of Activating transcription factor-2 (ATF2), a transcription factor of the ATF/CREB family, whereas the phosphorylation of Thr71 is constitutive and remains unchanged. The increased phosphorylation of ATF2 on Thr69 by JNK in the MPTP mouse model suggests a functional relationship between the transcriptional activation of ATF2 and dopaminergic neuron death. By using dopaminergic neuron-specific conditional ATF2 mutant mice, we found that either partial or complete deletion of the ATF2 DNA-binding domain in dopaminergic neurons markedly alleviates the MPTP-induced dopaminergic neurodegeneration, indicating that the activation of ATF2 plays a detrimental role in neuropathogenesis in PD. Taken together, our findings demonstrate that JNK-mediated ATF2 activation contributes to dopaminergic neuronal death in an MPTP model of PD. PMID:26515688

  7. A JNK-mediated autophagy pathway that triggers c-IAP degradation and necroptosis for anticancer chemotherapy.

    PubMed

    He, W; Wang, Q; Srinivasan, B; Xu, J; Padilla, M T; Li, Z; Wang, X; Liu, Y; Gou, X; Shen, H-M; Xing, C; Lin, Y

    2014-06-01

    Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin-1. Importantly, suppression of autophagy, with either pharmacological inhibitors or small interfering RNAs targeting the essential autophagy components ATG7 and Beclin-1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance. PMID:23831571

  8. Musca domestica larva lectin induces apoptosis in BEL-7402 cells through a Ca(2+)/JNK-mediated mitochondrial pathway.

    PubMed

    Wang, Chun-Ling; Xia, Yan; Nie, Jian-Zeng; Zhou, Minghui; Zhang, Rong-Ping; Niu, Li-Li; Hou, Li-Hua; Cao, Xiao-Hong

    2013-06-01

    Although Musca domestica larvae lectin (MLL) is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism(s) responsible for these processes remain elusive. In the current study, the signaling network underlying the MLL-induced apoptosis of human hepatoma BEL-7402 cell was investigated. Our data found out that MLL causes a sustained increase of the intracellular Ca(2+) and this process was prevented by the intracellular calcium chelator, BAPTA-AM, suggesting the involvement of intracellular Ca(2+) in MLL-induced cell apoptosis. MLL also causes the production of reactive oxygen species and elevates the phosphorylation status of JNK, processes associated with the increased cytoplasmic Ca(2+). The mitochondrial permeability transition pore (MPTP) opening study showed that MLL treatment of BEL-7402 cells results in the opening of MPTP and a reduction of mitochondrial transmembrane potential. In such condition, cytochrome-c was detected to be released from mitochondria to cytoplasm through the MPTP. This eventually activates caspase-3 and thus results in apoptosis of the tested BEL-7402 cells. According to a comprehensive review of all the evidence, it is concluded that MLL induces apoptosis of BEL-7402 cells through a Ca(2+)/JNK-mediated MPTP pathway. PMID:23247835

  9. Isothiocyanates inhibit the invasion and migration of C6 glioma cells by blocking FAK/JNK-mediated MMP-9 expression.

    PubMed

    Lee, Chang-Su; Cho, Hyun-Ji; Jeong, Yun-Jeong; Shin, Jae-Moon; Park, Kwan-Kyu; Park, Yoon-Yub; Bae, Young-Seuk; Chung, Il-Kyung; Kim, Mihyun; Kim, Cheorl-Ho; Jin, Fansi; Chang, Hyeun-Wook; Chang, Young-Chae

    2015-12-01

    Isothiocyanates (ITCs) derived from cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit preventative effects against various types of cancers. Yet, the inhibitory effects of ITCs on C6 glioma cell invasion and migration have not been reported. Thus, we aimed to analyze ITC-regulated MMP-9 activation, a crucial enzyme of cancer metastasis that degrades the extracellular matrix, in C6 glioma cells to investigate the inhibitory effects on cancer invasion and migration by ITCs. In the present study, we found that ITCs specifically suppressed PMA-induced MMP-9 secretion and protein expression. The inhibitory effects of ITCs on PMA-induced MMP-9 expression were found to be associated with the inhibition of MMP-9 transcription levels through suppression of nuclear translocation of NF-?B and activator protein-1 (AP-1). It was also confirmed that ITCs decreased MMP-9-mediated signaling such as FAK and JNK, whereas they had no effect on the phosphorylation of ERK and p38. Moreover, wound-healing and ?ranswell invasion assays showed that ITCs inhibited the migration and invasion of C6 glioma cells. These results suggest that ITCs could be potential agents for the prevention of C6 glioma cell migration and invasion by decreasing FAK/JNK-mediated MMP-9 expression. PMID:26397194

  10. Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways

    PubMed Central

    Yang, Chuan-bin; Pei, Wei-jing; Zhao, Jia; Cheng, Yuan-yuan; Zheng, Xiao-hui; Rong, Jian-hui

    2014-01-01

    Aim: To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms. Methods: Human breast cancer cell line MCF-7 and other tumor cell lines (T47D, HepG2, HeLa, and PC12) were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential (MMP) was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species (ROS) were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis. Results: Bornyl caffeate (10, 25, and 50 ?mol/L) suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 ?mol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), z-VAD (pan-caspase inhibitor) or the thiol antioxidant L-NAC. Conclusion: Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways. PMID:24335836

  11. Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

    SciTech Connect

    Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng; Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 ; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae; Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305; Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756

    2012-03-15

    Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

  12. Human pregnancy serum inhibits interleukin-2 production.

    PubMed Central

    Nicholas, N S; Panayi, G S; Nouri, A M

    1984-01-01

    Cell-mediated immunity may be depressed during pregnancy. We used the two way mixed lymphocyte reaction as an in vitro model of cell mediated immunity and studied the effect of pregnancy sera on this system by the amount of tritiated thymidine taken up by activated lymphocytes. We found that: (1) pregnancy sera contain a factor inhibiting the mixed lymphocyte reaction; (2) the inhibition of the mixed lymphocyte reaction induced by sera could be reversed by the addition of the supernatant from allogeneic mixed lymphocyte reaction; (3) pure interleukin-1 could not reverse the inhibitory effect and (4) recombinant interleukin-2 (IL-2) completely reversed the inhibitory effect of pregnancy sera on the mixed lymphocyte reaction. We conclude that a factor (or factors) present in serum from pregnant women is capable of inhibiting the generation of IL-2 during lymphocyte activation. PMID:6239719

  13. Interleukin-2: Biology, Design and Application.

    PubMed

    Arenas-Ramirez, Natalia; Woytschak, Janine; Boyman, Onur

    2015-12-01

    Interleukin-2 (IL-2) exerts crucial functions during immune homeostasis via its effects on regulatory T (Treg) cells, and the optimizing and fine-tuning of effector lymphocyte responses. Thus, somewhat paradoxically, low doses of recombinant IL-2 have been used for Treg cell-based immunosuppressive strategies against immune pathologies, while high-dose IL-2 has shown some success in stimulating anti-tumor immune responses. Recent studies of the functional, biophysical and structural characteristics of IL-2 have led to the generation of IL-2 formulations, including IL-2/mAb complexes and IL-2 variants (muteins) that selectively enhance IL-2's immune stimulatory versus inhibitory properties. Here, we review these findings, placing new mechanistic insights into improved next-generation IL-2 formulations within the broader context of IL-2 biology. We conclude by integrating these findings into a framework for understanding IL-2-mediated selective immune modulation. PMID:26572555

  14. Structural and functional characterisation of ferret interleukin-2.

    PubMed

    Ren, Bin; McKinstry, William J; Pham, Tam; Newman, Janet; Layton, Daniel S; Bean, Andrew G; Chen, Zhenjun; Laurie, Karen L; Borg, Kathryn; Barr, Ian G; Adams, Timothy E

    2016-02-01

    While the ferret is a valuable animal model for a number of human viral infections, such as influenza, Hendra and Nipah, evaluating the cellular immune response following infection has been hampered by the lack of a number of species-specific immunological reagents. Interleukin 2 (IL-2) is one such key cytokine. Ferret recombinant IL-2 incorporating a C-terminal histidine tag was expressed and purified and the three-dimensional structure solved and refined at 1.89 Å by X-ray crystallography, which represents the highest resolution and first non-human IL-2 structure. While ferret IL-2 displays the classic cytokine fold of the four-helix bundle structure, conformational flexibility was observed at the second helix and its neighbouring region in the bundle, which may result in the disruption of the spatial arrangement of residues involved in receptor binding interactions, implicating subtle differences between ferret and human IL-2 when initiating biological functions. Ferret recombinant IL-2 stimulated the proliferation of ferret lymph node cells and induced the expression of mRNA for IFN-γ and Granzyme A. PMID:26472619

  15. Human eosinophils express functional interleukin 2 receptors.

    PubMed Central

    Rand, T H; Silberstein, D S; Kornfeld, H; Weller, P F

    1991-01-01

    Because T cell-derived cytokines may affect the functioning of eosinophils, we have investigated the capacity of human eosinophils to respond to IL-2. IL-2 was a potent chemoattractant with ED50 of 10(-12) M with eosinophils from all normal and eosinophilic donors tested. The monoclonal antibodies anti-Tac and TU27 against p55 (Tac/CD25) and p75 receptor subunits, respectively, each inhibited IL-2-dependent eosinophil migration. The molar potency of IL-2 and the inhibitory activity of each of the above antibodies suggest that high affinity heterodimeric IL-2 receptor complexes mediated the migration responses of eosinophils to IL-2. Binding of monoclonal antibody against p75 was not detectable by flow cytometry, and high affinity binding sites for 125I-IL-2 were below the limits of quantitation on eosinophils from individuals with eosinophilia. Expression of p55 (Tac/CD25) by eosinophils, without requirement for in vitro activation, was demonstrable by flow cytometry, radioimmunoprecipitation, and Northern blotting for mRNA. Surface expression of p55 on eosinophils from normal or eosinophilic individuals increased during culture for 24-48 h with a biologic activity purified from stimulated U937 cells and to a lesser extent with granulocyte-macrophage CSF or lymphocyte chemoattractant factor but not with nine other cytokines. These studies indicate that blood eosinophils respond to IL-2 and identify one mechanism whereby activation of T lymphocytes may influence the function of eosinophils. Images PMID:1885772

  16. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

    SciTech Connect

    Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.; Sattabongkot, Jetsumon; Beerheide, Walter; Boelsterli, Urs A. . E-mail: phcbua@nus.edu.sg

    2006-11-15

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.

  17. Benzo[a]pyrene diol epoxide stimulates an inflammatory response in normal human lung fibroblasts through a p53 and JNK mediated pathway

    PubMed Central

    Dreij, Kristian; Rhrissorrakrai, Kahn; Gunsalus, Kristin C.; Geacintov, Nicholas E.

    2010-01-01

    Cellular responses to carcinogens are typically studied in transformed cell lines, which do not reflect the physiological status of normal tissues. To address this question, we have characterized the transcriptional program and cellular responses of human lung WI-38 fibroblasts upon exposure to the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE). In contrast to observations in cell lines, we find that BPDE treatment induces a strong inflammatory response in these normal fibroblasts. Whole-genome microarrays show induction of numerous inflammatory factors, including genes that encode interleukins (ILs), growth factors and enzymes related to prostaglandin synthesis and signaling. Real-time reverse transcriptionpolymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) revealed a time- and dose-dependent-induced expression and production of cyclooxygenase 2, prostglandin E2 and IL1B, IL6 and IL8. In parallel, cell cycle progression and DNA repair processes were repressed, but DNA damage signaling was increased via p53-Ser15 phosphorylation and induced expression levels of GADD45A, CDKN1A, BTG2 and SESN1. Network analysis suggested that activator protein 1 transcription factors may link the cell cycle response and DNA damage signaling with the inflammatory stressresponse in these cells. We confirmed this hypothesis by showing that p53-dependent signaling through c-jun N-terminal kinase (JNK) led to increased cJun-Ser63 phosphorylation and that inhibition of JNK-mediated cJun activation using p53- or JNK-specific inhibitors significantly reduced IL gene expression and subsequent production of IL8. This is the first demonstration that a strong inflammatory response is triggered in normal fibroblasts by BPDE and that this occurs through coordinated regulation with other cellular processes. PMID:20382639

  18. Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures.

    PubMed Central

    Arzt, E; Stelzer, G; Renner, U; Lange, M; Mller, O A; Stalla, G K

    1992-01-01

    The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells. Images PMID:1331177

  19. Soluble interleukin-2 receptor, interleukin-2 and interleukin-4 in sera and supernatants from patients with progressive systemic sclerosis.

    PubMed Central

    Famularo, G; Procopio, A; Giacomelli, R; Danese, C; Sacchetti, S; Perego, M A; Santoni, A; Tonietti, G

    1990-01-01

    We studied the sera of patients with progressive systemic sclerosis (PSS) for elevated levels of soluble interleukin-2 receptor (sIL-2R), interleukin-2 (IL-2) and interleukin-4 (IL-4). We also measured IL-2, IL-4 and B cell growth factor (BCGF) activity in supernatants of peripheral blood mononuclear cells from the same patients. The finding of elevated serum sIL-2R and IL-2, and the increased levels of IL-2, IL-4 and BCGF activity in culture supernatants indicates that T lymphocyte hyperactivity likely play a major role in PSS. The failure to detect under our experimental conditions a direct proliferative effect of recombinant IL-2 on enriched normal B cells might suggest that IL-4 is the cytokine mainly responsible of the BCGF activity recovered in PSS supernatants. PMID:2397608

  20. Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-?B Pathway: A Mouse Cardiomyocyte Model

    PubMed Central

    Xie, Zulong; Yang, Shuang; Xu, Wei; Hou, Jingbo; Yu, Bo

    2014-01-01

    Ginsenoside Rb3 is extracted from the plant Panax ginseng and plays important roles in cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. NF-?B is an important transcription factor involved in I/R injury. However, the underlying mechanism of ginsenoside Rb3 in myocardial I/R injury remains poorly understood. In the current study, a model of myocardial I/R injury was induced via oxygen and glucose deprivation (OGD) followed by reperfusion (OGD-Rep) in mouse cardiac myoblast H9c2 cells. Our data demonstrate that ginsenoside Rb3 suppresses OGD-Rep-induced cell apoptosis by the suppression of ROS generation. By detecting the NF-?B signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-?B activity. Ginsenoside Rb3 inhibits the upregulation of phospho-I?B-? and nuclear translocation of NF-?B subunit p65 which are induced by ORD-Rep injury. In addition, the extract also inhibits the OGD-Rep-induced increase in the expression of inflammation-related factors, such as IL-6, TNF-?, monocyte chemotactic protein-1 (MCP-1), MMP-2 and MMP-9. However, LPS treatment alleviates the protective roles of ginsenoside Rb3 and activates the NF-?B pathway. Finally, the upstream factors of NF-?B were analyzed, including the Akt/Foxo3a and MAPK signaling pathways. We find that ginsenoside Rb3 pretreatment only decreases the phosphorylation of JNK induced by OGD-Rep injury, an indicator of the MAPK pathway. Importantly, an inhibitor of phospho-JNK, SP600125, protects against OGD-Rep induced apoptosis and inhibited NF-?B signaling pathway, similar to the roles of ginsenoside Rb3. Taken together, our results demonstrate that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is attributed to the inhibition of JNK-mediated NF-?B activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury. PMID:25084093

  1. Interleukin 2 production in patients with thyroid diseases

    SciTech Connect

    Malaitsev, V.V.; Bogdanova, I.M.; Shatsova, E.N.

    1986-10-01

    Interleukin 2 (IL2) activity was determined in clinically healthy persons as well as in patients with various thyroid diseases. The state of thyroid function was assessed by radioimmunoassay. IL-2 activity was assessed by its ability to maintain proliferation of T blast cells. The T blast cells were obtained from mouse spleen cells. After incubation, /sup 3/H-thymidine was added to the prepared cells. The results of the three series of experiments performed are shown. It is concluded from the results that thyroid diseases are accompanied by changes in IL 2 production.

  2. Interleukin-2 therapy reverses some immunosuppressive effects of skeletal unloading

    NASA Technical Reports Server (NTRS)

    Armstrong, Jason W.; Balch, Signe; Chapes, Stephen K.

    1994-01-01

    Using antiorthostatic suspension, we characterized hematopoietic changes that may be responsible for the detrimental effect of skeletal unloading on macrophage development. Skeletally unloaded mice had suppressed macrophage development in unloaded and loaded bones, which indicated a systemic effect. Bone marrow cells from unloaded mice secreted less macrophage colony-stimulating factor and interleukin-6 than control mice. Additionally, T-lymphocyte proliferation was reduced after skeletal unloading. We show that polyethylene glycol-interleukin-2 therapy reversed the effects of skeletal unloading on macrophage development and cell proliferation.

  3. A humanized antibody that binds to the interleukin 2 receptor.

    PubMed Central

    Queen, C; Schneider, W P; Selick, H E; Payne, P W; Landolfi, N F; Duncan, J F; Avdalovic, N M; Levitt, M; Junghans, R P; Waldmann, T A

    1989-01-01

    The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac. Images PMID:2513570

  4. Interleukin-2 gene transfer into human transitional cell carcinoma of the urinary bladder

    PubMed Central

    Milella, M; Jacobelli, J; Cavallo, F; Guarini, A; Velotti, F; Frati, L; Fo, R; Forni, G; Santoni, A

    1999-01-01

    Transitional cell carcinoma of the bladder is one of the human cancers most responsive to immunotherapy, and local interleukin-2 (IL-2) production appears to be an important requirement for immunotherapy to be effective. In this study, we engineered two human bladder cancer cell lines (RT112 and EJ) to constitutively release human IL-2 by retroviral vector-mediated gene transfer. Following infection and selection, stable and consistent production of biologically active IL-2 was demonstrated at both the mRNA and the protein level. Morphology, in vitro growth rate and proliferation, as well as other cytokine gene mRNA or membrane adhesion receptor expression, were not altered in IL-2 transduced cells as compared to their parental or control vector-infected counterparts. Moreover, IL-2 engineered cells lost their tumorigenicity into nu/nu mice and the mechanism of rejection appeared to involve multiple host effector cell populations, among which a prominent role was played by neutrophils and radiosensitive cells. These findings may offer support to the development of an IL-2-based gene therapy approach to human bladder cancer. 1999 Cancer Research Campaign PMID:10070868

  5. Effect of spaceflight on lymphocyte proliferation and interleukin-2 production

    NASA Technical Reports Server (NTRS)

    Nash, Patricia V.; Konstantinova, Irina V.; Fuchs, Boris B.; Rakhmilevich, Alexandr L.; Lesniak, A. T.; Mastro, Andrea M.

    1992-01-01

    In this study, inguinal lymp node lymphocytes from rats flown on the Cosmos 2044 mission were tested for proliferation and interleukin-2 (IL-2) production. Cells cultured with mitogenic lectins, phorbol ester, and calcium ionophore, or T-cell mitogen and lymphokine, were assayed for DNA synthesis by (H-3) thymidine incorporation. Lymphocytes incubated with a T-cell mitogen alone also were tested for IL-2 production. Proliferation of lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from control and flown rats produced similar amounts of IL-23. Thus microgravity may act on lymphocytes in a tissue-specific manner, a new finding that could impact on the evaluation of spaceflight effects on immunocompetence.

  6. Expression of interleukin 2 receptors by monocytes from patients with acquired immunodeficiency syndrome and induction of monocyte interleukin 2 receptors by human immunodeficiency virus in vitro.

    PubMed Central

    Allen, J B; McCartney-Francis, N; Smith, P D; Simon, G; Gartner, S; Wahl, L M; Popovic, M; Wahl, S M

    1990-01-01

    A population of circulating mononuclear cells from patients with AIDS was identified which expressed interleukin 2 receptors (IL-2R). By dual-fluorescence flow microfluorometry, the patients' IL-2R+ cells were further identified as Leu M3+ monocytes (29.4 +/- 5.2% of the Leu M3+ cells were IL-2R+, n = 15), whereas Leu M3+ monocytes from normal subjects were IL-2R negative (2.0 +/- 0.42%; P less than 0.001). By Northern analysis, monocytes from AIDS patients, but not control subjects, constitutively expressed steady-state levels of IL-2R mRNA. Functionally, the IL-2R+ monocytes were capable of depleting IL-2 from culture supernatants, suggesting a mechanism for the reduced IL-2 levels commonly seen in AIDS patients. IL-2R+ monocytes also expressed increased levels of surface HLA-DR which may favor monocyte T-cell interactions and the transmission of human immunodeficiency virus (HIV). In additional studies, normal monocytes were infected with a macrophage-tropic HIV isolate in vitro and monitored for IL-2R and HLA-DR expression. Within 24-48 h after exposure to HIV in vitro, but before evidence of productive infection, greater than 25% of the monocytes became IL-2R+ with increasing numbers of IL-2R+ cells and HLA-DR levels through day 6. These early signaling effects of HIV could be mimicked by adding purified HIV envelope glycoprotein gp120 to the monocytes. This stimulation of monocytes before or independent of productive infection of the cells by HIV is consistent with in vivo observations of activated and/or abnormal functions by monocytes that do not appear to be infected with HIV in AIDS patients. Images PMID:2295695

  7. Interleukin 2 signaling involves the phosphorylation of Stat proteins.

    PubMed

    Frank, D A; Robertson, M J; Bonni, A; Ritz, J; Greenberg, M E

    1995-08-15

    One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. PMID:7544001

  8. Detection of salivary interleukin-2 in recurrent aphthous stomatitis

    PubMed Central

    Kalpana, R; Thubashini, M; Sundharam, B Sivapatha

    2014-01-01

    Objective: The present study was undertaken to estimate and compare salivary interleukin-2 (IL-2) levels in patients with recurrent aphthous stomatitis, among healthy controls and their variation with age and sex. Study Design: Saliva was collected from 60 patients within the age range of 16-60 years which included 30 patients (17 Females and 13 Males) with recurrent aphthous stomatitis and healthy control group consisted of 30 participants (18 Females and 12 Males). IL-2 estimation was done in both the groups using enzyme linked immunosorbent assay (ELISA). Statistical analysis of the data was done using Independent t test. Results: The results showed increased salivary IL-2 levels in patients with recurrent aphthous stomatitis compared to the healthy controls. The IL-2 levels were also increased in patients with the age group of 16-30 years compared to other age groups. Similar increase of IL-2 was also seen in female patients. Conclusion: Age related and sex related alterations of IL-2 in recurrent aphthous stomatitis patients were observed. PMID:25948989

  9. Intravenous ascorbic acid as an adjuvant to interleukin-2 immunotherapy

    PubMed Central

    2014-01-01

    Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. PMID:24884532

  10. Interleukin 2 exerts autocrine stimulation on murine T-cell leukaemia growth.

    PubMed Central

    Waldner, C. I.; Mongini, C.; Alvarez, E.; Sánchez Lockhart, M.; Gravisaco, M. J.; Hajos, S. E.

    1997-01-01

    As it has been suggested that an autocrine mechanism may control tumour cell growth, in this work cells from a spontaneous murine T lymphocyte leukaemia (LB) expressing the interleukin-2 receptor (IL-2R) (CD25) were evaluated in vitro for IL-2-mediated autocrine growth. Cells grew readily in culture and proliferation was enhanced by the addition of recombinant IL-2 but inhibited by monoclonal antibodies against either IL-2 or IL-2 receptor, in the absence of exogenous IL-2. Cyclosporin A also inhibited LB cell growth. However, when exogenous IL-2 was added together with cyclosporin A, cell proliferation proved similar to controls. Using reverse transcription polymerase chain reaction (PCR), mRNA for IL-2 was found to be present in tumour cells. Our findings support the hypothesis that LB tumour cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. Images Figure 5 PMID:9083328

  11. Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis.

    PubMed

    Fichna, Marta; ?urawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Za?uska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy

    2015-03-01

    Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p?=?0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p?=?0.004 and p?=?0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p?=?0.031 and p?=?0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p?=?0.036) and positively with serum antibodies to 21OH (p?=?0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p?=?0.022) and soluble IL2Ra secretion (p?=?0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61??4.3 versus 1.71??3.2?pg/mL, p?interleukin-2 and sIL2Ra. PMID:25347332

  12. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

    PubMed

    Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

    1996-02-15

    Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast. PMID:8769948

  13. Myelostimulatory activity of recombinant human interleukin-2 in mice

    SciTech Connect

    Talmadge, J.E.; Schneider, M.; Keller, J.; Ruscetti, F.; Longo, D.; Pennington, R.; Bowersox, O.; Tribble, H.

    1989-05-01

    In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.

  14. Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.

    PubMed Central

    Zmuidzinas, A; Mamon, H J; Roberts, T M; Smith, K A

    1991-01-01

    To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication. Images PMID:1708096

  15. The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta

    SciTech Connect

    Boehm, K.D.; Kelley, M.F.; Ilan, J.; Ilan, J. )

    1989-01-01

    The lymphokine interleukin 2 is an important immune system regulatory glycopolypeptide. It is produced by antigen- or mitogen-stimulated T lymphocytes and is required for the proliferation or clonal expansion of activated T lymphocytes. In this report, it is demonstrated by RNA transfer blot hybridization that the poly(A){sup +} RNA population of the human placenta contains a 0.85-kilobase RNA transcript that specifically hybridizes to a human interleukin 2 cDNA probe. By using hybridization histochemistry in situ, it is further shown that interleukin 2 RNA transcripts are localized, primarily, to the syncytial (syncytiotrophoblast) layer of the human placenta. Possible roles for syncytiotrophoblast-produced interleukin 2 are suggested and discussed.

  16. Transfection of murine myeloma cells to produce a chimeric antibody to the interleukin-2 receptor.

    PubMed

    Meng, Y G; Wong, T

    1994-10-21

    Murine myeloma X63Ag8.653 cells were transfected with heavy and light-chain expression vectors for a chimeric antibody (Ab) to the human interleukin-2 receptor. A cell line producing low quantities of the chimeric Ab was obtained and was transfected with either the cytomegalovirus (CMV) immediate-early gene ie1 or Epstein-Barr virus (EBV) BMLF1 DNA, together with the hygromycin B resistance (HyR) encoding gene for selection to improve productivity. Two cell lines with a four to eightfold increase in productivity were obtained. They showed higher levels of heavy- and light-chain mRNA expression. CMV ie1 or EBV BMLF1 DNA was not detected and no integration pattern changes for the heavy- and light-chain DNA were seen. The long-term productivity of one of the cell lines showed hygromycin B (Hy) requirement. Transfection with the HyR DNA alone also resulted in cells with increased productivity. The expression vectors contained the immunoglobulin light-chain enhancer kappa B DNA sequences (kappa B site). Nuclear extracts from parent myeloma cells showed one kappa B-binding protein band on a polyacrylamide gel, but nuclear extracts from transfected cells showed two additional slower-migrating bands. Increased Ab production correlated with an increased ratio of the medium-mobility kappa B-binding protein band to the high-mobility band. The possibility that Hy used for selection activated kappa B-binding proteins and increased Ab expression is discussed. PMID:7958965

  17. Chemical Modification of Recombinant Interleukin 2 by Polyethylene Glycol Increases Its Potency in the Murine Meth A Sarcoma Model

    NASA Astrophysics Data System (ADS)

    Katre, Nandini V.; Knauf, Michael J.; Laird, Walter J.

    1987-03-01

    Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life. This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol. The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and isoelectric focusing. This conjugate was compared to unmodified recombinant interleukin 2 in vitro and in vivo. Covalent attachment of the hydrophilic polymer polyethylene glycol enhanced the solubility of interleukin 2, decreased its plasma clearance, and increased its antitumor potency in the Meth A murine sarcoma model.

  18. Dehydroepiandrosterone triggers autophagic cell death in human hepatoma cell line HepG2 via JNK-mediated p62/SQSTM1 expression.

    PubMed

    Vegliante, Rolando; Desideri, Enrico; Di Leo, Luca; Ciriolo, Maria Rosa

    2016-03-01

    Autophagy is a catabolic process that cancer cells usually exploit during stress conditions to provide energy by recycling organelles and proteins. Beyond its prosurvival role, it is well accepted that occurrence of autophagy is often associated with a particular type of programmed cell death known as autophagic cell death (ACD). Dehydroepiandrosterone (DHEA) is an endogenous hormone showing anticancer properties even if the underlying mechanisms are not fully clear yet. Here, we provide evidence that DHEA induces ACD in human hepatoma cell line, HepG2. Indeed, autophagy inhibitors (i.e. 3-methyladenine or Atg5 siRNA) significantly reduced the percentage of dead cells. DHEA induces p62-dependent autophagy, which turns detrimental and brings about death. DHEA stimulates reactive oxygen species-independent jun N-terminal kinase (JNK) phosphoactivation and the treatment with JNK inhibitor reduces p62 mRNA levels, as well as DHEA-induced ACD. The transcription factor nuclear factor (erythroid-derived-2)-like-2 (Nrf2) constitutes the link between JNK and p62 since its migration to the nucleus is suppressed by JNK inhibitor and its inhibition through a dominant negative Nrf2 plasmid transfection decreases p62 protein levels. Overall, our data indicate that DHEA induces ACD in HepG2 via a JNK-Nrf2-p62 axis. Thus, DHEA could represent a new appealing drug for eliminating tumor cells through autophagy particularly in apoptosis-resistant cases. PMID:26762228

  19. Functional interleukin-2 receptors on intestinal epithelial cells.

    PubMed Central

    Ciacci, C; Mahida, Y R; Dignass, A; Koizumi, M; Podolsky, D K

    1993-01-01

    The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa. Images PMID:8326018

  20. In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

    NASA Technical Reports Server (NTRS)

    Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

    1996-01-01

    Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

  1. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design.

    PubMed

    Waldmann, Thomas A

    2006-08-01

    Interleukin-2 and interleukin-15 have pivotal roles in the control of the life and death of lymphocytes. Although their heterotrimeric receptors have two receptor subunits in common, these two cytokines have contrasting roles in adaptive immune responses. The unique role of interleukin-2 is in the elimination of self-reactive T cells to prevent autoimmunity. By contrast, interleukin-15 is dedicated to the prolonged maintenance of memory T-cell responses to invading pathogens. As discussed in this Review, the biology of these cytokines will affect the development of novel therapies for malignancy and autoimmune diseases, as well as the design of vaccines against infectious diseases. PMID:16868550

  2. 77 FR 22283 - Availability of an Environmental Assessment for Field Testing Feline Interleukin-2...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-13

    ... Animal and Plant Health Inspection Service Availability of an Environmental Assessment for Field Testing Feline Interleukin-2 Immunomodulator, Live Canarypox Vector AGENCY: Animal and Plant Health Inspection... assess the potential effects of this product on the safety of animals, public health, and the...

  3. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes

    PubMed Central

    Preston, Gavin C; Sinclair, Linda V; Kaskar, Aneesa; Hukelmann, Jens L; Navarro, Maria N; Ferrero, Isabel; MacDonald, H Robson; Cowling, Victoria H; Cantrell, Doreen A

    2015-01-01

    Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses. PMID:26136212

  4. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

    PubMed

    Preston, Gavin C; Sinclair, Linda V; Kaskar, Aneesa; Hukelmann, Jens L; Navarro, Maria N; Ferrero, Isabel; MacDonald, H Robson; Cowling, Victoria H; Cantrell, Doreen A

    2015-08-01

    Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses. PMID:26136212

  5. Purification and refolding of Escherichia coli-expressed recombinant human interleukin-2.

    PubMed

    Esfandiar, Samaneh; Hashemi-Najafabadi, Sameereh; Shojaosadati, Seyed Abbas; Sarrafzadeh, Shokuh Aazam; Pourpak, Zahra

    2010-04-01

    The expression of rhIL-2 (recombinant human interleukin-2) in bacteria results in the formation of insoluble inclusion-body aggregates. These aggregates were first solubilized under denaturing conditions (sodium phosphate buffer solution containing 8 M urea and 10 mM 2-mercaptoethanol) and then purified using IMAC (immobilized metal-ion-affinity chromatography). IMAC was used to capture rhIL-2. The protein was gradually refolded on the column by a gradient elution (8 M to 0 M urea) in the presence of 10% (v/v) glycerol. Glycerol was used to prevent protein aggregation during the refolding step. Using this method, rhIL-2 was collected at 97% purity and its activity was measured by the lymphocyte transformation test. The measured activity was identical with commercial human interleukin-2. PMID:20236092

  6. Human Immune Disorder Arising from Mutation of the ? Chain of the Interleukin-2 Receptor

    NASA Astrophysics Data System (ADS)

    Sharfe, Nigel; Dadi, Harjit K.; Shahar, Michal; Roifman, Chaim M.

    1997-04-01

    Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor ? chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

  7. Radioiodination of interleukin 2 to high specific activities by the vapor-phase chloramine T method

    SciTech Connect

    Siekierka, J.J.; DeGudicibus, S.

    1988-08-01

    Recombinant human interleukin 2 (IL-2) was radioiodinated utilizing the vapor phase chloramine T method of iodination. The method is rapid, reproducible, and allows the efficient radioiodination of IL-2 to specific activities higher than those previously attained with full retention of biological activity. IL-2 radioiodinated by this method binds with high affinity to receptors present on phytohemagglutinin-stimulated peripheral blood lymphocytes and should be useful for the study of receptor structure and function.

  8. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression.

    PubMed Central

    June, C H; Ledbetter, J A; Gillespie, M M; Lindsten, T; Thompson, C B

    1987-01-01

    CD28 is a homodimeric glycoprotein expressed on the surface of a major subset of human T cells that has recently been identified as a member of the immunoglobulin supergene family. The binding of monoclonal antibodies to the CD28 antigen on purified T cells does not result in proliferation; however, previous studies have shown that the combination of CD28 stimulation and protein kinase C activation by phorbol myristate acetate (PMA) results in T-cell proliferation that is independent of both accessory cells and activation of the T-cell receptor-CD3 complex. In the present study, effects of stimulation by anti-CD28 on cell cycle progression and on the interleukin 2 (IL-2) and IL-2 receptor system have been investigated on primary cultures of purified peripheral-blood CD28+ T cells. There was no measurable effect on cell size or on DNA synthesis after stimulation of resting (G0) cells by CD28 alone. After 3 h of activation of T cells by PMA alone, a slight (8%) increase in cell volume occurred that did not progress to DNA synthesis. In contrast, T-cell stimulation by CD28 in combination with PMA resulted in a progressive increase in cell volume in approximately 100% of cells at 12 to 14 h after stimulation. Northern blot (RNA blot) analysis revealed that CD28 stimulation alone failed to cause expression of the alpha chain of the IL-2 receptor or of IL-2 mRNA, and in accord with previous studies, stimulation by PMA alone resulted in the accumulation of IL-2 receptor transcripts but no detectable IL-2 mRNA. In contrast, T-cell stimulation by the combination of CD28 and PMA resulted in the appearance of IL-2 transcripts and enhanced expression of IL-2 receptor mRNA. Functional studies revealed that the proliferation induced by CD28 and PMA stimulation was entirely resistant to cyclosporine, in contrast to T-cell activation induced by the CD3-T-cell receptor complex. Cyclosporine was found not to affect the accumulation of IL-2 mRNA after CD28 plus PMA stimulation, although there was no detectable IL-2 mRNA after stimulation by CD3 in the presence of the drug. Furthermore, stimulation by CD28 in combination with immobilized CD3 antibodies caused a striking enhancement of IL-2 mRNA expression that was, in part, resistant to the effects of cyclosporine. These studies indicate that the CD28 molecule synergizes with protein kinase C activation to induce IL-2 gene expression and demonstrate that stimulation by the CD28 pathway can cause vigorous T-cell proliferation even in the presence of cyclosporine and that cyclosporine does not prevent transcription of 16-2 mRNA, as has been suggested previously. Moreover, these findings suggest that a potential role for the CD28 molecule in vivo may be to augment IL-2 production after stimulation of the CD3-T-cell receptor molecular complex and thereby to amplify an antigen-specific immune response. Finally, these results provide further evidence that the CD28 molecule triggers T-cell proliferation in a manner that differs biochemically from CD3-T-cell receptor-induced proliferation. Images PMID:2830495

  9. Soluble interleukin-2 receptors in serum and urine of patients with chancroid and their response to therapy.

    PubMed

    Abeck, D; Korting, H C; Zaba, R; Dangor, Y; Fehler, G; Ballard, R C

    1990-07-01

    To investigate cell-mediated immune response in chancroid, soluble interleukin-2 receptor levels in serum and urine samples of healthy individuals and patients were measured by an enzyme-linked immunosorbent assay. Increased levels both in serum and in urine were observed in cases of Haemophilus ducreyi infection. In patients showing a prolonged incubation period, urine levels exceeded serum values. Therapy led to a reduction of elevated interleukin-2 receptor levels in serum and in urine. PMID:2088539

  10. Effects of interleukin-2 on bioelectric activity of rat atrial myocardium under normal conditions and during gradual stretching.

    PubMed

    Aksyonov, A; Mitrokhin, V M; Mladenov, M I

    2015-09-01

    Using micro-electrode technique we studied the effects of interleukin-2 (50 ng/ml) on bio-electric activity of rat atrial myocardium under normal conditions and after gradual stretching of the tissue. It was shown that interleukin-2 caused increasing in the duration of action potential at the levels of 25, 50, and 90% re-polarization. Perfusion with interleukin-2 resulted in appearance of frequent rhythm patterns followed by smooth transient fragments of paroxysmal tachyarrhythmia pacing into normal rhythms. In the presence of interleukin-2, stretching of the tissue by 1.7 mN led to appearance of abnormal bio-electrical activity, predominantly in the lengthening of the duration of action potential at the levels of 90% re-polarization. Close observation of both interleukin-2 induced action potential duration to 90% of re-polarization, hump-like depolarization and stretch induced hump-like alteration, indicate existence of a link between the interleukin-2 and stretch induced mechanisms. PMID:26112420

  11. Adoptive Immunotherapy of Established Pulmonary Metastases with LAK Cells and Recombinant Interleukin-2

    NASA Astrophysics Data System (ADS)

    Mule, James J.; Shu, Suyu; Schwarz, Susan L.; Rosenberg, Steven A.

    1984-09-01

    The activation of human peripheral blood leukocytes or murine splenocytes with interleukin-2 (IL-2) generated cells that were lytic in vitro for a variety of fresh tumor cells. The adoptive transfer of such lymphokine-activated killer (LAK) cells to mice with established pulmonary sarcoma metastases was highly effective in reducing the number (and size) of these tumor nodules when combined with repeated injections of recombinant IL-2. These findings provide a rationale for clinical trials of the infusion of human LAK cells generated with recombinant IL-2 as well as Phase I trials of the infusion of recombinant IL-2 systemically into humans.

  12. Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor beta chain does not contribute to internalization of interleukin-2.

    PubMed

    Furse, R K; Malek, T R

    1993-12-01

    To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) alpha- and beta-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2R beta cDNA led to surface expression of IL-2R beta and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the beta subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated beta cDNA, several CX1 transfectants were produced that expressed a beta-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type beta-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the beta subunit and raise the possibility that such signals may be entirely contained within the gamma subunit. PMID:8258333

  13. Effect of human colostrum on interleukin-2 production and natural killer cell activity.

    PubMed Central

    Sirota, L.; Straussberg, R.; Notti, I.; Bessler, H.

    1995-01-01

    The effect of human colostrum on the production of interleukin-2 (IL-2) and on natural killer (NK) cell activity by peripheral blood mononuclear cells (PBMC) was investigated in 50 healthy women. At concentrations as low as 0.5%, human colostrum stimulated IL-2 production; at a higher concentration (10%), IL-2 secretion was inhibited. A time and dose dependent inhibitory effect of colostrum on NK cytotoxicity was also observed. This inhibition could be reversed by the addition of human recombinant IL-2 (hrIL-2). The stimulation of IL-2 production induced by human colostrum might compensate for its inhibitory effect on NK cell activity. These findings suggest an additional mechanism by which breast feeding may affect the neonatal immune system. PMID:7583615

  14. Anti-interleukin-2 receptor antibody (daclizumab) treatment of corticosteroid-refractory autoimmune thrombocytopenic purpura.

    PubMed

    Fogarty, Patrick F; Seggewiss, Ruth; McCloskey, Donna Jo; Boss, Carol A; Dunbar, Cynthia E; Rick, Margaret E

    2006-02-01

    We administered daclizumab, a humanized monoclonal anti-interleukin-2 receptor (IL-2R) antibody, to 11 patients with corticosteroid-refractory autoimmune thrombocytopenic purpura (AITP) every 2 weeks for five treatments. Of nine evaluable patients, one individual experienced a partial response. Lymphocyte phenotyping by flow cytometry indicated post-treatment binding of IL-2Ra by daclizumab in all patients. Mid-study serum soluble IL-2R levels in all patients increased 4-15 -fold over baseline values (p=0.004). Despite these measurable immunologic effects, blockade of the IL-2/IL-2R axis did not effectively abrogate the autoimmune response in this group of patients with corticosteroid-refractory AITP. PMID:16461323

  15. Interleukin-2 at the Crossroads of Effector Responses, Tolerance, and Immunotherapy

    PubMed Central

    Liao, Wei; Lin, Jian-Xin; Leonard, Warren J.

    2013-01-01

    Interleukin-2 is a pleiotropic cytokine produced after antigen activation that plays pivotal roles in the immune response. Discovered as a T-cell growth factor, IL-2 additionally promotes CD8+ T cell and NK cell cytolytic activity, and modulates T cell differentiation programs in response to antigen, promoting nave CD4+ T cell differentiation into T helper-1 (Th1) and T helper-2 (Th2) cells while inhibiting T helper-17 (Th17) and T follicular helper (Tfh) cell differentiation. Moreover, IL-2 is essential for the development and maintenance of T regulatory (Treg) cells and for activation-induced cell death, thereby mediating tolerance and limiting inappropriate immune reactions. In this review, we focus on the molecular mechanisms and complex cellular actions of IL-2, its cooperative and opposing effects with other cytokines, and how both promoting and blocking the actions of IL-2 are being utilized in clinical medicine. PMID:23352221

  16. Use of enhanced interleukin-2 formulations for improved immunotherapy against cancer.

    PubMed

    Rosalia, Rodney A; Arenas-Ramirez, Natalia; Bouchaud, Grgory; Raeber, Miro E; Boyman, Onur

    2014-12-01

    The use of interleukin-2 (IL-2) for the stimulation of an effector immune response against metastatic cancer dates back to the early 1980s. Administration of unmodified IL-2, either alone or together with antigen-specific approaches, has resulted in remarkably long-term survival of some patients suffering from metastatic melanoma. However, such treatment is usually hampered by the appearance of toxic adverse effects, which has motivated the engineering of modified IL-2 formulations showing reduced toxicity while being more potent at stimulating anti-tumor effector immune cells. In this review we summarize and discuss the features and biological relevance of several enhanced IL-2 formulations, compare these to IL-15-based therapeutics, and try to foreshadow their potential in immunological research and immunotherapy. PMID:25271022

  17. Effects of anti-Tac antibody on the response of large granular lymphocytes to interleukin-2.

    PubMed Central

    Mukaida, N; Kasahara, T; Hosoi, J; Shioiri-Nakano, K; Kawai, T

    1986-01-01

    Large granular lymphocytes (LGL), which consist of a unique population comprising almost all of the natural killer (NK) cell activity, were separated from peripheral blood mononuclear cells by sequential depletion of monocytes and conventional discontinuous Percoll density gradient sedimentation. The LGL populations thus obtained exhibited significant levels of interferon-gamma (IFN-gamma) production and proliferation, as well as augmentation of NK cell activity in response to interleukin-2 (IL-2). Among these IL-2-driven phenomena, only IFN-gamma production was markedly inhibited by the monoclonal anti-Tac antibody, which presumably recognized the IL-2 receptor, whereas the proliferative response and the augmentation of NK cell activity were only minimally affected by the same antibody, even at the higher concentration. The dissociation of the effects of anti-Tac antibody on these IL-2-driven events gives us some insight on the role of IL-2 receptors involved in these immunologically interesting functions of IL-2. PMID:3002967

  18. Interleukin 2 receptor expression by human blood lymphocytes after vaccination with pneumococcal polysaccharides.

    PubMed

    Tvede, N; Heilmann, C; Christensen, L D

    1989-06-01

    Proliferative responses of unseparated peripheral blood mononuclear cells (PBMC) and blood T cells to recombinant interleukin 2 (rIL-2) were significantly increased 7-21 days after the vaccination with pneumococcal polysaccharides (PPS). In contrast, non-T cells expressed increased responsiveness to rIL-2 only on post-vaccination day 7. Analysis of the proliferative response to rIL-2 among lymphocyte subsets (CD4+Leu8+, CD4+Leu8-, CD8+Leu8+, CD8+Leu8-, CD20+) in cultures of unseparated PBMC revealed that the CD8+Leu8- T cells expressed increased responsiveness 7-14 days after vaccination, whereas neither CD4+ (Leu8+ and Leu8-) nor CD8+Leu8+ T cells showed significantly increased responsiveness after vaccination. The CD20+ B cells, like non-T cells, expressed increased responsiveness to rIL-27 days after the vaccination only. Expression of the 55 kD low-affinity interleukin 2 receptor (IL-2R, CD 25) on freshly isolated PBMC, as judged by direct fluorescence staining with a MoAb anti-55 kD chain, was low (less than 3%) and an increased expression of this receptor was not detected following vaccination. In contrast, binding of 125I-labelled IL-2 to freshly isolated PBMC increased following vaccination (day 7). Scatchard plot analysis revealed a modest increase in the expression of high-affinity IL-2R (Kd = 1-2 pM), whereas the increase in expression of the 75-kD, intermediate-affinity IL-2R (Kd = 300 pM) was more pronounced (from 195 to 295 (means) receptors per PBMC). It is concluded that, following vaccination with PPS increased IL-2R expression is induced on blood lymphocytes. These investigations suggest a role for T cells in the human immune response against PPS. PMID:2787716

  19. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

    PubMed Central

    Andersson, M; Gunne, H; Agerberth, B; Boman, A; Bergman, T; Sillard, R; Jrnvall, H; Mutt, V; Olsson, B; Wigzell, H

    1995-01-01

    A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells. Images PMID:7737114

  20. The p75 peptide is the receptor for interleukin 2 expressed on large granular lymphocytes and is responsible for the interleukin 2 activation of these cells.

    PubMed Central

    Tsudo, M; Goldman, C K; Bongiovanni, K F; Chan, W C; Winton, E F; Yagita, M; Grimm, E A; Waldmann, T A

    1987-01-01

    There are at least two interleukin 2 (IL-2) binding peptides: one is the Mr 55,000 peptide (p55) reactive with the anti-Tac monoclonal antibody, and the other is a Mr 75,000 non-Tac IL-2 binding peptide (p75). Independently existing Tac or p75 peptides represent low-affinity IL-2 receptors, whereas high-affinity IL-2 receptors are expressed when both peptides are present and associated in a receptor complex. It has long been known that normal large granular lymphocytes (LGL) or leukemic cells from the patients with abnormal expansions of LGL can be activated by IL-2 not only to more-potent natural killer cells but also to effectors of lymphokine-activated killer (LAK) activity, although they do not express the Tac peptide. In the present study, using cross-linking methodology, we found that normal LGL and leukemic LGL from all individuals tested expressed the p75 IL-2 binding peptide but did not express the Tac peptide. These LGL leukemia cells made proliferative responses to IL-2 but required a much higher concentration than that required for the proliferation of normal phytohemagglutinin-stimulated T lymphoblasts that express high-affinity receptors. Furthermore, the addition of IL-2 to Tac-negative LGL leukemic cells augmented transcription of the Tac gene and induced the Tac peptide. Neither the IL-2-induced proliferation nor the up-regulation of Tac gene expression was inhibited by the addition of anti-Tac. These results strongly suggest that the p75 peptide is responsible for IL-2-induced activation of LGL and that the p75 peptide alone can mediate an IL-2 signal. Thus, the p75 peptide may play an important role in the IL-2-mediated immune response not only by participating with the Tac peptide in the formation of the high-affinity receptor complex on T cells but also by contributing to the initial triggering of LGL activation so that these cells become efficient natural killer and lymphokine-activated killer cells. Images PMID:3110786

  1. Soluble Interleukin 2 Receptor Levels, Temperament and Character in Formerly Depressed Suicide Attempters Compared with Normal Controls

    ERIC Educational Resources Information Center

    Rothenhausler, Hans-Bernd; Stepan, Alexandra; Kapfhammer, Hans-Peter

    2006-01-01

    An imbalance of the immune system and mixed personality profiles in suicide attempters have been reported. As suicidal behavior is common in patients with psychiatric disorders within the spectrum of depressive features, in this study we measured soluble interleukin-2 receptor concentrations in plasma (sIL-2R) and investigated temperament and

  2. Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant Newcastle disease virus (rNDV) has shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tu...

  3. Soluble Interleukin 2 Receptor Levels, Temperament and Character in Formerly Depressed Suicide Attempters Compared with Normal Controls

    ERIC Educational Resources Information Center

    Rothenhausler, Hans-Bernd; Stepan, Alexandra; Kapfhammer, Hans-Peter

    2006-01-01

    An imbalance of the immune system and mixed personality profiles in suicide attempters have been reported. As suicidal behavior is common in patients with psychiatric disorders within the spectrum of depressive features, in this study we measured soluble interleukin-2 receptor concentrations in plasma (sIL-2R) and investigated temperament and…

  4. Administration of high-dose continuous infusion interleukin-2 to patients age 70 or over.

    PubMed

    Quan, Walter; Ramirez, Maria; Taylor, Chris; Quan, Francine; Vinogradov, Mikhail; Walker, Paul

    2005-02-01

    High-dose bolus or continuous infusion interleukin-2-based therapy can cause capillary leak syndrome. Significant cardiovascular/hemodynamic events, including myocardial infarction, hypotension, pulmonary edema, and cardiac arrhythmia, have been described with such therapy. Concern over the toxicity of highdose interleukin-2 (IL-2) therapy has led to some clinicians excluding patients 70 years of age or over. We have treated 15 patients 70 years of age or over having an Eastern Conference Oncology Group (ECOG) performance status of 0 or 1, with therapy based on continuous infusion IL-2 18 MIU/sq m/24 hours for 72 hours. All patients underwent a pretreatment evaluation of cardiac status with a low-level stress or adenosine stress test. Cycles were typically repeated every 3 weeks for 4 cycles, then every 3-4 weeks thereafter. Patients were treated by oncology nurses in either the stem cell transplant (intermediate unit) or the oncology inpatient unit. Patient characteristics were: median age, 72 years (range, 70-83 years); tumor types: melanoma (10), kidney cancer (5); most common sites of disease: lung (11), lymph nodes (6), subcutaneous (3), liver (2); prior therapy included: none (8), outpatient IL-2 (5), other immunotherapy (4). Median number of cycles received: 3 (1-10). Most common toxicities were: fever, rigors, nausea, emesis, hypophosphatemia, and hypomagnesemia. Three patients required the use of dopamine for blood pressure support. Two patients declined further therapy. There were no treatment-related deaths. No patients required endotracheal intubation or transfer to an intensive care unit. One complete and 8 partial responses (60% response rate) have been seen. Responding sites include the lung, lymph node, intact kidney primary, and liver. Median survival has not been reached at over 14 months (range 3+-26+ months). Patients who are 70 years of age and older with an ECOG performance status of 0 or 1 are able to tolerate high-dose continuous infusion IL-2-based therapy and may respond to such treatment. PMID:15778574

  5. Biochemical and immunological properties of rat recombinant interleukin-2 and interleukin-4.

    PubMed Central

    McKnight, A J; Classon, B J

    1992-01-01

    We have previously described the isolation and sequencing of cDNA clones encoding rat interleukin-2 (IL-2) and interleukin-4 (IL-4). In the present study, we report the generation of stably transfected Chinese hamster ovary (CHO) cell lines which constitutively synthesize and secrete high levels of rat recombinant IL-2 (rIL-2) and IL-4 (rIL-4). The expression of the cytokine cDNA sequences is driven by the human cytomegalovirus promoter/enhancer within the respective pEE6. HCMV-GS vector constructs, following the successful transfection and isolation of methionine sulphoximine (MSX)-resistant CHO cell lines. Analyses of metabolically labelled CHO.rIL-2 and CHO.rIL-4 have been performed, in addition to studies which demonstrate certain biological properties of these recombinant cytokines including T-cell growth factor activity (rIL-2) and the ability to enhance expression of class II major histocompatibility complex (MHC) molecules on spleen cells (rIL-4). The availability of large quantities of these rat recombinant cytokines, conveniently produced by a mammalian cell line, will prove invaluable in future studies into the induction and regulation of immune responses in this species. Images Figure 2 Figure 5 Figure 6 PMID:1551691

  6. Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2).

    PubMed

    Shih, C Y; Truitt, R L; Andreani, M; Bortin, M M

    1983-10-01

    Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity. PMID:6605296

  7. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro

    SciTech Connect

    Rubin, L.A.; Kurman, C.C.; Fritz, M.E.; Biddison, W.E.; Boutin, B.; Yarchoan, R.; Nelson, D.L.

    1985-11-01

    With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2R by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.

  8. Expression of nine-banded armadillo (Dasypus novemcinctus) interleukin-2 in E. coli.

    PubMed

    Adams, J E; Pea, M T; Gillis, T P; Williams, D L; Adams, L B; Truman, R W

    2005-12-01

    The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type leprosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression. The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lymphoblasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of immunological tools will assist in advancing the armadillo as a translational model for leprosy. PMID:16338142

  9. Identification of a Cytotoxic Form of Dimeric Interleukin-2 in Murine Tissues

    PubMed Central

    Wrenshall, Lucile E.; Clabaugh, Suzanne E.; Cool, David R.; Arumugam, Prakash; Grunwald, William C.; Smith, Deandra R.; Liu, Gino C.; Miller, John D.

    2014-01-01

    Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include “traditional” IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems. PMID:25019288

  10. Interleukin 2 activity in chronic liver disease and the effect of in vitro alpha-interferon.

    PubMed Central

    Saxena, S; Nouri-Aria, K T; Anderson, M G; Eddleston, A L; Williams, R

    1986-01-01

    To investigate mitogen induced helper interleukin 2 (IL-2) production in patients with chronic liver disease (CLD), IL-2 activity was assessed by an IL-2 bioassay using phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMNC). IL-2 activity was significantly reduced in patients with autoimmune chronic active hepatitis, primary biliary cirrhosis and alcoholic hepatitis with or without cirrhosis (P less than 0.01), and was comparable to controls in those with alcoholic cirrhosis alone. In vitro preincubation of PBMNC with lymphoblastoid alpha-interferon (alpha-IFN) before stimulation with PHA, led to a significant increase in IL-2 activity in all subjects (P less than 0.01), except those with alcoholic hepatitis, but in none of the groups did the levels of IL-2 activity reach those seen in normal subjects. The decrease in IL-2 activity in patients with CLD may be due to low IL-2 production or presence of an IL-2 antagonist(s). Such an abnormality may occur, not only as a result of liver damage, but may also be important in determining immunological disturbances involved in the pathogenesis of the liver disease. PMID:3486732

  11. Interleukin-2, interleukin-12, and interferon-γ levels and risk of young adult Hodgkin lymphoma

    PubMed Central

    Gill, Parkash S.; Salam, Muhammad T.; Nieters, Alexandra; Masood, Rizwan; Cockburn, Myles G.; Gauderman, W. James; Martínez-Maza, Otoniel; Nathwani, Bharat N.; Pike, Malcolm C.; Van Den Berg, David J.; Hamilton, Ann S.; Deapen, Dennis M.; Mack, Thomas M.

    2008-01-01

    Young adult Hodgkin lymphoma (YAHL) is associated clinically with altered immunity, including a systemic defect in cell-mediated responses. There is strong evidence of a genetic contribution to risk, so we hypothesized that heritable alterations in cytokine production associated with Th1 function may contribute to susceptibility. We identified twin pairs in whom at least one member had YAHL and measured interleukin-2 (IL-2), interleukin-12 (IL-12), and interferon-γ (IFN-γ) levels in PHA-stimulated peripheral blood mononuclear cell supernatant in 90 case-twins, 84 of their disease-free twins (unaffected cotwins), and 90 matched controls. Mean difference and mean percentage difference in cytokine levels between case-twins and controls, and unaffected cotwins and controls were determined using analysis of covariance. YAHL case-twins and their unaffected cotwins had IL-12 levels that were 60.6% (P = .002) and 49% (P = .04) lower than those of their matched controls, respectively. IL-2 levels were significantly higher in case-twins (P = .049), but not unaffected cotwins (P = .57), compared with controls. Differences in IFN-γ levels were not statistically significant in either comparison. An IL-12 polymorphism known to regulate expression was associated with a 2.8-fold (P = .03) increase in YAHL risk. Thus, both case-twins and their unaffected cotwins had a decreased ability to produce IL-12, which may contribute to YAHL susceptibility. PMID:18077789

  12. Role of Interleukin-2 in Uremic Pruritus Among Attendants of AL-Zahraa Hospital Dialysis Unit

    PubMed Central

    Azim, Amira Adel Abdel; Farag, Asmaa Saied; El-Maleek Hassan, Doaa Abd; Abdu, Safaa Mahmoud Ismail; Lashin, Somaya Mohamed Abo-Elfetouh; Abdelaziz, Nahla Mohamed

    2015-01-01

    Background: Uremic pruritus (UP) is a very distressing symptom and remains one of the most frustrating and potentially disabling symptoms in patients with end-stage renal disease (ESRD). Its etiopathogenesis remains unclear and complex. The aim of this study was to investigate the possible role of interleukin-2 (IL-2) in UP, and correlate its level with the severity of itching in ESRD patients. Patients and Methods: This study was carried out on 60 patients on maintenance hemodialysis (HD), 30 patients with UP and 30 patients without UP, and 30 apparently healthy age- and sex-matched subjects as controls. Itch intensity was scored as mild, moderate, and severe using five-dimensional itch scale. Some relevant clinical parameters (age, sex, xerosis, presence of neuropathy, duration of dialysis, complete medical history, and history of pruritic skin diseases) and laboratory findings including creatinine, urea, calcium, phosphorus, parathyroid hormone, and serum levels of IL-2 were evaluated. Results: In our study, we found a statistically significant difference in IL-2 level between patients and controls. However, there was no statistically significant difference in IL-2 levels between cases with pruritus and cases without pruritus. Also, there was a statistically significant relation between IL-2 level and duration of the disease. Conclusion: Further studies are needed to understand the contribution of IL-2 and possibly other cytokines in the pathogenesis of this distressing symptom in ESRD. PMID:25814728

  13. Selectively targeting an inactive conformation of interleukin-2-inducible T-cell kinase by allosteric inhibitors.

    PubMed

    Han, Seungil; Czerwinski, Robert M; Caspers, Nicole L; Limburg, David C; Ding, WeiDong; Wang, Hong; Ohren, Jeffrey F; Rajamohan, Francis; McLellan, Thomas J; Unwalla, Ray; Choi, Chulho; Parikh, Mihir D; Seth, Nilufer; Edmonds, Jason; Phillips, Chris; Shakya, Subarna; Li, Xin; Spaulding, Vikki; Hughes, Samantha; Cook, Andrew; Robinson, Colin; Mathias, John P; Navratilova, Iva; Medley, Quintus G; Anderson, David R; Kurumbail, Ravi G; Aulabaugh, Ann

    2014-06-01

    ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays. PMID:24593284

  14. Evidence for tumor reduction in refractory or relapsed B-CLL patients with infusional interleukin-2.

    PubMed

    Kay, N E; Oken, M M; Mazza, J J; Bradley, E C

    1988-01-01

    Recombinant interleukin-2 (rIL-2) is a biologic response modifier that is capable of enhancing or restoring the cytolytic capacity of large granular lymphocytes (LGL). We utilized this biologic response modifier in the treatment of B-chronic lymphocytic leukemia (B-CLL), a disease frequently characterized by deficient or absent natural killer activity. B-CLL (n = 12) patients previously refractory to chemotherapy or with progressive disease post cessation of chemotherapy were eligible. rIL-2 was given as i.v. infusion (2 x 10(6) units/m2) over 2 h 5 times per week for 3 weeks as induction. Responding patients were placed on maintenance therapy. Although there were no complete or partial responses (by ECOG criteria) there was clear evidence of tumor reduction. Seven of 10 evaluable patients had a reduction of the peripheral blood B cell clone, 3 had node reduction and 2 had reduction in their splenomegaly. All patients experienced mild to moderate toxicity and 1 patient died while on induction therapy. Three B-CLL patients following induction rIL-2 treatment were placed back on chemotherapy because of progressive disease. Interestingly, these 2 B-CLL patients achieved extremely rapid and complete responses to chemotherapy which had previously been ineffective. These data suggest a possible role for rIL-2 in treatment of B-CLL. PMID:3265509

  15. The development of new immunotherapies for the treatment of cancer using interleukin-2. A review.

    PubMed Central

    Rosenberg, S A

    1988-01-01

    Recent increases in knowledge of cellular immunology, combined with developments in biotechnology, have provided new opportunities for the development of immunotherapies for the treatment of cancer in humans. One approach to therapy is that of adoptive immunotherapy, that is, the transfer to the tumor bearing host of lymphoid cells with antitumor reactivity that can mediate antitumor responses. Several lymphocyte subpopulations have now been identified that may be suitable for use in adoptive immunotherapy. Resting lymphocytes incubated in interleukin-2 (IL-2) give rise to lymphokine activated killer (LAK) cells that can lyse malignant cells, but not normal cells. Clinical studies in patients with advanced cancer have revealed that treatment with high dose IL-2 alone or in combination with LAK cells can mediate the complete or partial regression of cancer in selected patients. Other approaches are currently undergoing investigation, including the adoptive transfer of tumor infiltrating lymphocytes, which, in animal models, have antitumor reactivity 50-100 times more potent than do LAK cells. Other new approaches to immunotherapy include the use of combination of lymphokines, such as the use of tumor necrosis factor or alpha interferon in conjunction with IL-2. The availability of recombinant lymphokines that provide large amounts of biologically active materials can hopefully lead to the development of effective new therapies for cancer in humans. Images Fig. 4. Fig. 5. Figs. 6A and B. Fig. 7. Figs. 8A and B. Fig. 9. Fig. 10. Fig. 11. Figs. 12A and B. Figs. 13. Fig. 14. Fig. 15. PMID:3041925

  16. Adhesion molecule expression and leucocyte trafficking following immunotherapy with recombinant interleukin-2.

    PubMed

    Miles, D W; Happerfield, L C; Bobrow, L G; Rubens, R D

    1996-04-01

    The relevant anti-tumour mechanisms of recombinant interleukin-2 (rIL-2) in vivo are unclear but an influx of T-lymphocytes and macrophages has been noted in regressing lesions. One of the dose limiting toxicities of rIL-2 is the development of a capillary leak syndrome attributed to widespread endothelial activation. Changes in expression of endothelial and leucocyte-associated adhesion molecules were assessed in tumour and uninvolved skin in patients with metastatic breast cancer receiving rIL-2. Increased expression of intracellular adhesion molecule-1, its leucocyte-associated ligand, leucocyte function associated molecule-1, vascular cell adhesion molecule and its ligand, very late after activation antigen-4 as well as members of the selectin family of adhesion molecules, were noted in uninvolved skin following rIL-2. Expression of these adhesion molecules was noted in tumour stroma before rIL-2 but little change was observed following rIL-2 infusion. An influx of monocytes and T-lymphocytes (expressing the IL-2 receptor and of the memory subtype) and a lower number of neutrophils was noted in uninvolved skin following rIL-2. Although monocytes and T-lymphocytes were present in tumour stroma before rIL-2 no changes were observed following infusion. The changes noted in the dermis contrast with those seen at tumour sites and may partly explain the low therapeutic index of rIL-2. PMID:8732338

  17. Interleukin-2-Dependent Allergen-Specific Tissue-Resident Memory Cells Drive Asthma.

    PubMed

    Hondowicz, Brian D; An, Dowon; Schenkel, Jason M; Kim, Karen S; Steach, Holly R; Krishnamurty, Akshay T; Keitany, Gladys J; Garza, Esteban N; Fraser, Kathryn A; Moon, James J; Altemeier, William A; Masopust, David; Pepper, Marion

    2016-01-19

    Exposure to inhaled allergens generates T helper 2(Th2) CD4(+) Tcells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) Tcells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memorycells in the lymphoid organs and tissue-residentmemory cells in the lung. Experimental blockade oflymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) Tcells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses. PMID:26750312

  18. High-dose interleukin 2 promotes bacterial translocation from the gut.

    PubMed Central

    Reynolds, J. V.; Murchan, P.; Leonard, N.; Gough, D. B.; Clarke, P.; Keane, F. B.; Tanner, W. A.

    1995-01-01

    Toxicity associated with high-dose recombinant interleukin 2 (rIL-2) therapy simulates a sepsis syndrome, but the mechanism remains unclear. We hypothesised that translocated gut-origin bacteria may be important. Fifty-one male rats were randomised to receive rIL-2 by intraperitoneal injection at doses (IU) of 10(5) (n = 15), 10(4) (n = 8), 10(3) (n = 8) or 10(2) (n = 8) twice daily, or a saline bolus (n = 12). After 5 days, ileal histomorphology was assessed and the mesenteric lymph node complex cultured. Results showed that colonisation of mesenteric lymph nodes with Escherichia coli occurred in all rats treated with 10(5) IU of rIL-2, and in 62%, 37% and 12% of rats treated with decreasing doses of rIL-2. No translocation was observed in control animals. An increase in submucosal lymphatics and occasional mucosal disruption was seen only in the group receiving 10(5) IU. These data show that rIL-2 promotes bacterial translocation and suggests a mechanism that may fuel high-dose rIL-2 toxicity in man. Images Figure 1 PMID:7669573

  19. Interleukin-2 Expression in Lupoid and Usual Types of Old World Cutaneous Leishmaniasis

    PubMed Central

    Mashayekhi Goyonlo, Vahid; Elnour, Hesameldin; Nordlind, Klas

    2014-01-01

    Background: Interleukin (IL)-2 plays a central role in T cell-dependent immune responses. Objectives: We conducted this study to determine and compare IL-2 expression in lupoid and usual types of Old World Cutaneous Leishmaniasis (OWCL), using immunohistochemistry. Patients and Methods: Thirteen paraffin-embedded specimens of lupoid and 12 specimens of usual types of OWCL were used. A mouse monoclonal anti IL-2 antibody was used for staining by the envision technique. Results: There were strongly stained discrete foci of staining through inflammatory infiltrates of dermis and also in basal layers of epidermis and adnexal structures, with a distinctive pattern of hot spot activity foci (mean of 9.31 6.4 versus 8.17 6.9 foci per HPF for lupoid and usual types, respectively). The expression of IL-2 had no correlation with the pattern of granulomatous inflammation (tuberculoid, sarcoidal or mixed suppurative). Conclusions: Interleukin-2 takes part in the immunological response of the granulomatous reaction of OWCL and is not statistically different between lupoid and usual types (P = 0.674). PMID:25763226

  20. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    PubMed Central

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-? (IFN-?) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-? gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-? and NF-AT. PMID:21435270

  1. Interleukin 2 defect in the peripheral blood and the lung in patients with Sjgren's syndrome.

    PubMed Central

    Miyasaka, N; Murota, N; Yamaoka, K; Sato, K; Yamada, T; Nishido, T; Okuda, M

    1986-01-01

    We studied interleukin 2 (IL-2) production both in the peripheral blood and the lung in patients with Sjgren's syndrome (SS). IL-2 production of the peripheral blood mononuclear cells (PBMC) was significantly impaired in patients with SS (P less than 0.001). The patients with extraglandular disease and with associated connective tissue disease were more defective in IL-2 production. The defect could not be attributable to culture conditions. Both OKT4+ and OKT8+ T cells were deficient in producing IL-2. However, impaired IL-2 production could be partly restored by either (1) adding PMA to the PHA-stimulated culture, or (2) supplementing indomethacin (IM) from the initiation of the culture, or (3) depletion of adherent cells from PBMC. Furthermore, SS T cells were more sensitive to PGE1 than normal controls. In contrast, the response of PBMC to IL-2 was not disturbed in SS. IL-1 production of SS PBMC was not defective although there seemed to be suppressive factor(s) produced by SS adherent cells. In addition, IL-2 production of SS pulmonary lymphocytes was also decreased, suggesting that IL-2 producing cells might not be sequestrated in the lung. These data suggest that qualitative T cell defects and suppressor macrophages might be responsible for defective IL-2 production in SS and that IL-2 deficiency may contribute to the disordered immunoregulation in SS. PMID:3490935

  2. Interleukin 2 in the pathogenesis and therapy of type 1 diabetes.

    PubMed

    Rosenzwajg, Michelle; Churlaud, Guillaume; Hartemann, Agns; Klatzmann, David

    2014-12-01

    Regulatory T cells (Tregs) play a major role in controlling effector T cells (Teffs) responding to self-antigens, which cause autoimmune diseases. An improper Treg/Teff balance contributes to most autoimmune diseases, including type 1 diabetes (T1D). To restore a proper balance, blocking Teffs with immunosuppressants has been the only option, which was partly effective and too toxic. It now appears that expanding/activating Tregs with low-dose interleukin-2 (IL-2) could provide immunoregulation without immunosuppression. This is particularly interesting in T1D as Tregs from T1D patients are reported as dysfunctional and a relative deficiency in IL-2 production and/or IL-2-mediated signaling could contribute to this phenotype. A clinical study of low-dose IL-2 showed a very good safety profile and good Treg expansion/activation in T1D patients. This opens the way for efficacy trials to test low-dose IL-2 in prevention and treatment of T1D and to establish in which condition restoration of a proper Treg/Teff balance would be beneficial in the field of autoimmune and inflammatory diseases. PMID:25344788

  3. Construction and characterization of encapsidated poliovirus replicons that express biologically active murine interleukin-2.

    PubMed

    Basak, S; McPherson, S; Kang, S; Collawn, J F; Morrow, C D

    1998-05-01

    Poliovirus genomes have been constructed in which the capsid genes have been substituted with the murine gene encoding interleukin-2 (IL-2) (referred to as replicons). One replicon contained the gene for IL-2 in place of the poliovirus capsid VP2 and VP3 genes, and a second replicon was constructed that contained the murine IL-2 substituted for the poliovirus VP3 and VP1 genes. The IL-2 genes were cloned into the replicon so as to maintain the translational reading frame with the remaining poliovirus proteins. Transfection of either replicon into cells resulted in the expression of replicon-encoded proteins and replication of replicon RNA. Using a procedure developed in this laboratory, we have encapsidated these replicons into authentic polio virions by passaging the replicons in the presence of a recombinant vaccinia virus, VVP1, which expresses the capsid precursor, P1, protein. Using a quantitative immunoassay, we determined that the majority of the IL-2 produced remained intracellular, with approximately 1%-2% released from the infected cells, and that the IL-2 was biologically active. The results of these studies demonstrate the utility of poliovirus replicons for expression of small bioactive molecules and are discussed with respect to future applications as immune adjuvants as well as potential new tumor therapies. PMID:9620357

  4. Transcriptional Activation of the Interleukin-2 Promoter by Hepatitis C Virus Core Protein

    PubMed Central

    Bergqvist, Anders; Rice, Charles M.

    2001-01-01

    Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56lck, and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections. PMID:11134290

  5. beta. -Adrenergic receptor-mediated suppression of interleukin 2 receptors in human lymphocytes

    SciTech Connect

    Feldman, R.D.; Hunninghake, G.W.; McArdle, W.L.

    1987-11-15

    Adrenergic receptor agonists are know to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of ..beta..-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the ..beta..-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of ..beta..-adrenergic agonists on expression of the high affinity IL-2 receptors, (/sup 125/I)IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of ..beta..-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that ..beta..-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites.

  6. Synergism between alpha-interferon and interleukin-2-activated killer cells: in vitro studies.

    PubMed

    Di Raimondo, F; LaPushin, R; Hersh, E M

    1987-01-01

    The effects of a combination of recombinant alpha-interferon (IFN-alpha) and interleukin-2 (IL-2)-activated human killer cells (lymphokine-activated killer or LAK cells) on Hs294T (IFN-sensitive) and A375P (IFN-resistant) human melanoma cell lines were evaluated. Pretreatment of target cells with IFN-alpha for at least 1 day increased their susceptibility to the lytic activity of LAK cells. The combination of the two agents in sequence (IFN-alpha followed by LAK cells) resulted in a true synergystic killing of both IFN-alpha-sensitive and resistant tumor cells. No synergy was observed when the sequence was reversed (LAK cells followed by IFN-alpha). When peripheral blood mononuclear cells were incubated simultaneously with IFN-alpha and IL-2, LAK cell generation and antitumor activity was markedly inhibited when tested against both IFN-treated and -non-treated tumor cells. These studies may be used to plan clinical trials of combination cytokine therapy for human cancer. PMID:3124452

  7. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  8. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    SciTech Connect

    Cervia, Davide; Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano ; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  9. Recombinant interleukin 2-activated natural killer cells regulate IgG2a production.

    PubMed

    Amigorena, S; Bonnerot, C; Fridman, W H; Teillaud, J L

    1990-08-01

    In this report we have analyzed the effect of recombinant interleukin 2 (rIL 2) and rIL 2-activated natural killer (NK) cells on the production of immunoglobulin isotypes by lipopolysaccharide (LPS)-stimulated spleen cells from nude mice. We found that rIL 2 induced a dose-dependent increase of IgG2a secretion and a concomitant inhibition of the secretion of other Ig isotypes. The analysis of the phenotype of LPS- and LPS+ rIL 2-stimulated nude spleen cells showed the appearance of a Thy-1+ asialo GM-1+slgM-CD3-CD4-CD8- cell population in the presence of rIL 2. A population with a similar phenotype was generated upon stimulation of spleen cells from nude mice with rIL 2 alone. These cells lysed YAC-1 cells, did not contain the alpha or gamma transcripts encoding the corresponding T cell receptor chains and are therefore NK cells activated by rIL 2. In co-culture experiments, these cells selectively increased the secretion of IgG2a by LPS-stimulated splenocytes from nude mice. The IgG2a induction triggered by rIL 2-activated NK cells, as well as that triggered by rIL 2, were blocked by an anti-interferon gamma monoclonal antibody. Thus, rIL 2 activated-NK cells enhance the production of IgG2a by secreting interferon-gamma. PMID:1698633

  10. Exploiting a natural conformational switch to engineer an Interleukin-2 superkine

    PubMed Central

    Levin, Aron M.; Bates, Darren L.; Ring, Aaron M.; Krieg, Carsten; Lin, Jack T.; Su, Leon; Moraga, Ignacio L.; Raeber, Miro E.; Bowman, Gregory R.; Novick, Paul; Pande, Vijay S.; Fathman, C. Garrison; Boyman, Onur; Garcia, K. Christopher

    2012-01-01

    The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells13. Considerable effort has been invested using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary high affinity receptor complex consisting of IL-2, IL-2R? (termed CD25), IL-2R?, and ?c48. Nave T cells express only a low density of IL-2R? and ?c, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2R? and?c. Here, using in vitro evolution, we eliminated IL-2s functional requirement for CD25 expression by engineering an IL-2 superkine (termed super-2) with increased binding affinity for IL-2R?. Crystal structures of super-2 in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, including a flexible helix in the IL-2R? binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in super-2 recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation T cells irrespective of CD25 expression. Compared to IL-2, super-2 induced superior expansion of cytotoxic T cells, leading to improved anti-tumor responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary edema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy. PMID:22446627

  11. Molecular cloning of a functional bovine interleukin 2 cDNA.

    PubMed Central

    Reeves, R; Spies, A G; Nissen, M S; Buck, C D; Weinberg, A D; Barr, P J; Magnuson, N S; Magnuson, J A

    1986-01-01

    A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences. Images PMID:3486415

  12. Low-Dose Interleukin-2 Therapy: A Driver of an Imbalance between Immune Tolerance and Autoimmunity

    PubMed Central

    Kosmaczewska, Agata

    2014-01-01

    For many years, the role of interleukin-2 (IL-2) in autoimmune responses was established as a cytokine possessing strong pro-inflammatory activity. Studies of the past few years have changed our knowledge on IL-2 in autoimmune chronic inflammation, suggesting its protective role, when administered at low-doses. The disrupted balance between regulatory and effector T cells (Tregs and Teffs, respectively) is a characteristic of autoimmune diseases, and is dependent on homeostatic cytokines, including IL-2. Actually, inherent defects in the IL-2 signaling pathway and/or levels leading to Treg compromised function and numbers as well as Th17 expansion have been attributed to autoimmune disorders. In this review, we discuss the role of IL-2 in the pathogenesis of autoimmune diseases. In particular, we highlight the impact of the dysregulated IL-2 pathway on disruption of the Treg/Th17 balance, reversal of which appears to be a possible mechanism of the low-dose IL-2 treatment. The negative effects of IL-2 on the differentiation of follicular helper T cells (Tfh) and pathogenic Th17 cells, both of which contribute to autoimmunity, is emphasized in the paper as well. We also compare the current IL-2-based therapies of animal and human subjects with immune-mediated diseases aimed at boosting the Treg population, which is the most IL-2-dependent cell subset desirable for sufficient control of autoimmunity. New perspectives of therapeutic approaches focused on selective delivery of IL-2 to inflamed tissues, thus allowing local activity of IL-2 to be combined with its reduced systemic and pleiotropic toxicity, are also proposed in this paper. PMID:25322151

  13. Immunotherapy with Canarypox Vaccine and Interleukin-2 for HIV-1 Infection: Termination of a Randomized Trial

    PubMed Central

    Smith, Kendall A; Andjelic, Sofija; Popmihajlov, Zoran; Kelly-Rossini, Liza; Sass, Aquanette; Lesser, Martin; Benkert, Steven; Waters, Cory; Ruitenberg, Joyce; Bellman, Paul

    2007-01-01

    Objectives: To determine whether immunotherapy of chronic HIV-1 infection can prevent or attenuate viremia upon antiviral discontinuation. Design: This was a Phase II randomized, partially double blinded, 2×2 factorial study of three steps of 12 wk/step. Step I involved four groups: (1) vaccine placebo, (2) vaccine (ALVAC, vCP1452), (3) placebo + interleukin 2 (IL-2), and (4) vaccine + IL-2. Step II involved a 12-wk diagnostic treatment interruption (DTI). Step III involved an extension of the DTI for an additional 12 wk. Setting: The Weill-Cornell General Clinical Research Center. Participants: Chronically infected HIV-1 positive adults with undetectable HIV-1 levels and > 400 CD4+ T cells/μl. Interventions An HIV canarypox vaccine (vCP1452) and vaccine placebo, administered every 4 wk for four doses, and low-dose IL-2 administered daily for 12–24 wk. Outcome measures: Primary endpoints: (1) Proportion of participants with undetectable plasma HIV RNA during trial Step II, (2) mean log10 HIV RNA copies/ml ([HIV]) from weeks 21–25, and (3) proportion of individuals eligible for trial Step III. Results: 44 participants were randomized, but 16 withdrew or were withdrawn before completing Step II. As all participants underwent viral relapse in Step II, the study was terminated after 28 participants completed Step II. Among the four groups, there was no difference in mean [HIV] or the proportion of individuals with < log10 4.48 HIV; no difference between the mean [HIV] of the two groups that received ALVAC (n = 17) versus placebo (n = 11); and no significant difference between the mean [HIV] of the two groups that received IL-2 (n = 11) versus placebo (n = 17). Conclusions: Neither ALVAC (vCP1452) nor low-dose daily IL-2 nor their combination prevented the relapse of viremia upon discontinuation of antiviral therapy. PMID:17260026

  14. Administration in vivo of recombinant interleukin 2 protects mice against septic death.

    PubMed Central

    Weyand, C; Goronzy, J; Fathman, C G; O'Hanley, P

    1987-01-01

    Administration in vivo of recombinant interleukin 2 (rIL-2) to mice induces a polyclonal IgM response. When co-administered with a specific antigen, rIL-2 can enhance concentrations of murine IgM antibodies specific for the antigen by fivefold within 7 d of initial treatment. IgM antibodies that are induced after injection of rIL-2 include antibodies specific for J5, a cell wall core lipopolysaccharide (LPS) antigen that is shared by the different members of the Enterobactericeae family. We report here that mice pretreated with rIL-2 or immunized with J5 antigen 7 d before bacterial challenge were protected from septic death that is caused by intraperitoneal challenges with Escherichia coli. Optimal protection was provided by a combined J5 antigen and rIL-2 treatment. Acquisition of the rIL-2 and J5 antigen-induced protection against lethal bacterial infection coincided temporally with maximal serum IgM titers that also contained IgM antibodies specific for the J5 antigen. In passive immunization experiments, the affinity-purified IgM fraction in sera of rIL-2-treated animals was identified as necessary and sufficient for protection. The IgM-depleted serum had no protective effect. The nonspecific augmentation of host-defense mechanisms without the induction of endotoxin manifestations makes rIL-2 a potential candidate to any alternative LPS-containing vaccines for the prevention of bacterial infections by gram-negative organisms since the core LPS antigen is shared among gram-negative bacteria. PMID:3294901

  15. Phase I study of cancer therapy with recombinant interleukin-2 administered by intravenous bolus injection.

    PubMed

    Hersh, E M; Murray, J L; Hong, W K; Rosenblum, M G; Reuben, J M; Weilbaecher, R; Sarwal, A N; Bradley, E C; Konrad, M; Arnett, F C

    1989-01-01

    Sixty-six patients with disseminated malignancy were treated with recombinant interleukin-2 (IL-2) on a three times a week (M, W, F) IV-bolus injection schedule. Doses ranged from 0.001 to 14.0 x 10(6) units/M2 body surface area. Consecutive groups of 3-5 patients were placed on each dose level and were maintained on that level except for dosage de-escalation for toxicity. Toxicity to all major organ systems were noted with major toxicity including fever and chills, anorexia, fatigue and malaise, arthralgias and arthritis as well as hepatic and renal toxicity. All toxicity reversed within one week of drug cessation. Renal toxicity manifested by azotemia, arthritis and fatigue were the common dose limiting toxicities and the maximally tolerated dose was 12 x 10(6) units/M2. Pharmacokinetic studies indicated a short half-life (T1/2 alpha = 7-23 minutes). At doses over 0.5 x 10(6) units/M2 increases in absolute lymphocytes and eosinophil counts were noted. All T lymphocyte subsets increased. Maximal increases were seen at 4-8 x 10(6) units/M2 with a lesser increase at 10-14 x 10(6) units/M2 dosage level. Circulating NK cells also increased while circulating LAK cells were detected during therapy. Partial responses were noted in 3 patients with melanoma. These lasted 4, 6 and 16 months and involved pulmonary, pulmonary plus mesenteric and retro-orbital plus hepatic metastases respectively in these patients. PMID:2642025

  16. Interleukin 2 Topical Cream for Treatment of Diabetic Foot Ulcer: Experiment Protocol

    PubMed Central

    2015-01-01

    Background It is estimated there are 2.9 million diabetic patients in the United Kingdom, and around 5%-7% of patients have diabetic ulcers. This number will continue to increase globally. Diabetic ulcers are a major economic burden on the healthcare system. More than £650 million is spent on foot ulcers or amputations each year, and up to 100 people a week have a limb amputated due to diabetes. In T1DM, the level of IL-2 is reduced, and hence, wound healing is in a prolonged inflammatory phase. It is not known if IL-2 topical cream can shorten the healing process in T1DM patients. Objective The objective of this study is to understand the pathophysiology in type 1 diabetes (T1DM) and investigate possible future treatment based on its clinical features. The hypothesis is that IL-2 cream can speed up wound healing in NOD mice and that this can be demonstrated in a ten-week study. An experiment protocol is designed in a mouse model for others to conduct the experiment. The discussion is purely based on diabetic conditions; lifestyle influences like smoking and drinking are not considered. Methods Skin incisions will be created on 20 nonobese diabetic (NOD) mice, and IL-2 topical cream will be applied in a 10-week study to prove the hypothesis. Mice will be randomly and equally divide into two groups with one being the control group. Results T1DM patients have a decreased number of T regulatory (Treg) cells and interleukin 2 (IL-2). These are the keys to the disease progression and delay in wound healing. Diabetic ulcer is a chronic wound and characterized by a prolonged inflammatory phase. Conclusions If the experiment is successful, T1DM patients will have an alternative, noninvasive treatment of foot ulcers. In theory, patients with other autoimmune diseases could also use IL-2 topical cream for treatment. PMID:26276522

  17. Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice.

    PubMed Central

    Saxena, R K; Saxena, Q B; Adler, W H

    1984-01-01

    Generation of natural killer (NK) activity in response to a partially purified preparation of rat interleukin-2 (IL-2) was compared in spleen cells derived from young (8-10 weeks old) and old (greater than 2 years old) female C57BL/6 mice. Significant NK activation was observed in both young and old mouse spleen cells incubated with 100 U IL-2/ml for 1-4 days, but the levels of cytotoxic activity generated in old mouse spleen cells was always lower than those of similarly treated young mouse spleen cells. Differences in IL-2-induced NK activation in old and young mouse spleen cells was obtained irrespective of the concentration of IL-2 used (25-400 U/ml). Quantitative comparisons indicated that old spleen cells activated by 3 day incubation with IL-2 acquired about two-fold higher NK activity than fresh young mouse spleen cells but still had only one-fourth of the levels of NK activity attained by IL-2-activated young mouse spleen cells. Cytotoxic activity of IL-2-activated young or old mouse spleen cells were totally abrogated by anti-asialo GM-1 antiserum + C but not by anti-Ly-2 + C treatment, indicating that the activated cytotoxic cells fell in the NK cell category. An analysis of NK precursor (NK-p) frequency by limiting dilution assay indicated that the NK-p frequency was about 4-fold higher in young as compared to old mouse spleen cells. The level of cytotoxic activity attained per NK-p cell was not significantly different for NK-p cells of old or young mice. PMID:6608487

  18. Effect of combined treatment with recombinant interleukin-2 and allicin on pancreatic cancer.

    PubMed

    Wang, Cong-Jun; Wang, Chao; Han, Jiang; Wang, Yong-Kun; Tang, Lin; Shen, Dong-Wei; Zhao, Yi; Xu, Rong-Hua; Zhang, Hui

    2013-12-01

    This study aimed to evaluate the efficacy of combined treatment with recombinant interleukin-2 (rIL-2) and allicin on pancreatic cancer and explore the potential immunological mechanism. A total of 60 C57/BL6 nude mice pancreatic cancer xenograft models were randomized into four groups of 15 mice per group: control group, allicin treatment group, rIL-2 treatment group, combined treatment with allicin and rIL-2 group. Mice in each group were treated with saline, rIL-2, allicin, or combination of rIL-2 and allicin by weekly i.v injection for four weeks. After four weeks of treatment, eyeballs of the mice were extracted and blood was drawn, percentages of CD4+T, CD8+T and NK cell were analyzed by FACS, IFN-? level was detected by ELISA. One mouse in each group was sacrificed to measure the weight and volume of the tumor and prepared to the paraffin section of tumor tissue. Apoptosis of the tumor cells was analyzed by TUNEL and FACS. Other mice continued to receive treatment, survival period were compared between each group. We observed a significant suppression of xenograft growth and a significant prolonged survival time in the combined treatment with allicin and rIL-2 group (P<0.05). The most amount of apoptotic cells were observed in the combined therapy group (P<0.05). The percentages of CD4+T, CD8+T and NK cell and serum IFN-? level increased significantly in the combined treatment group compared with other groups (P<0.05). Combined treatment with allicin and rIL-2 resulted in suppression of tumor growth and prolonged survival time possibly through activation of CD4+T, CD8+T and NK cell. PMID:24135803

  19. Systemic Administration of Interleukin 2 Enhances the Therapeutic Efficacy of Dendritic Cell-Based Tumor Vaccines

    NASA Astrophysics Data System (ADS)

    Shimizu, K.; Fields, R. C.; Giedlin, M.; Mule, J. J.

    1999-03-01

    We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of non-toxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-? production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

  20. Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism

    PubMed Central

    Gründemann, Carsten; Thell, Kathrin; Lengen, Karin; Garcia-Käufer, Manuel; Huang, Yen-Hua; Huber, Roman; Craik, David J.; Schabbauer, Gernot; Gruber, Christian W.

    2013-01-01

    Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides. PMID:23840803

  1. Antigen-specific suppressor T cells respond to recombinant interleukin-2 and other lymphokines.

    PubMed Central

    Taylor, C E; Fauntleroy, M B; Stashak, P W; Baker, P J

    1991-01-01

    Previous studies have shown that transfer of whole spleen cell populations obtained from primed donors or transfer of purified T cells enriched for suppressor activity (Ts) to recipient mice decreased the antibody response to pneumococcal polysaccharide type III (SSS-III) when the animals were simultaneously immunized with SSS-III. In the present studies, such suppression of the antibody response was transferred with 10- to 100-fold fewer primed spleen cells when the cells were treated in vitro with recombinant interleukin-2 (rIL-2) before transfer; spleen cells from naive mice or mice primed with an unrelated antigen (dextran) and then treated with rIL-2 did not cause suppression of the antibody response to SSS-III, thereby eliminating the possibility of nonspecific carryover effects induced by rIL-2. In vivo administration of rIL-2 at the time of immunization with an optimally immunogenic dose of SSS-III resulted in significant (P less than 0.05) suppression of the antibody response relative to that of control animals, suggesting that IL-2 augments the clonal expansion of Ts cells in vivo. Further, the ability of passively administered anti-IL-2 receptor antibody to inhibit generation of Ts cells in vivo is consistent with such a view. Spleen cells from primed animals treated with rIL-4, rIL-5, or gamma interferon--but not those from primed animals treated with rIL-6--likewise were able to transfer suppression of the antibody response with fewer cells than those required when primed cells not treated with lymphokines were used. Thus, these studies indicate that Ts cell activity is greatly influenced by lymphokines produced by helper T cells. The studies also suggest that these lymphokines are required during activation and/or clonal expansion of Ts cells. PMID:1824762

  2. Leukemia-derived growth factor (non-interleukin 2) produced by a human malignant T lymphoid cell line.

    PubMed Central

    Uittenbogaart, C H; Fahey, J L

    1982-01-01

    A growth factor was found in the supernatants of MOLT-4f, a cell line derived from acute T lymphoblastic leukemia. This factor, which we designated leukemia-derived growth factor from MOLT-4f (LDGF-M4), is different from interleukin 2. LDGF-M4 has features of a polypeptide with a molecular weight in the range of 5,000-15,000, as indicated by gel diffusion chromatography. LDGF-M4 does stimulate MOLT-4f and at least two other T cell lines that do not respond to interleukin 2. Because MOLT-4f cells produce and respond to LDGF-M4, this factor may contribute to the independence of MOLT-4f and related T leukemia cell lines. PMID:6983693

  3. Elevated serum levels of neopterin and soluble interleukin-2 receptor in patients with ovarian cancer.

    PubMed

    Gadducci, A; Ferdeghini, M; Malagnino, G; Prontera, C; Fanucchi, A; Annicchiarico, C; Bianchi, R; Fioretti, P; Facchini, V

    1994-03-01

    Preoperative serum neopterin, soluble interleukin-2 receptor (sIL-2R), and CA125 levels were assayed in 47 patients with ovarian cancer and 113 patients with benign ovarian disease undergoing laparotomy. The cutoff limits of the antigens for the preoperative evaluation of ovarian cancer were fixed according to the Youden plot, using the patients with benign ovarian disease as controls. These limits were 7.9 nmole/liter for neopterin, 71 U/ml for sIL-2R, and 83 U/ml for CA125. The preoperative mean values of serum neopterin and sIL-2R were significantly higher in patients with ovarian cancer than in those with benign ovarian disease. Therefore these tests would seem to be useful in distinguishing benign from malignant ovarian masses. Serum levels of neopterin, sIL-2R, and CA125 above the cutoff limits were detected in 66.0, 78.7, and 76.6% of patients with ovarian cancer. Patients with advanced-stage disease (FIGO > or = III) were significantly more likely to have a higher percentage of elevated values of sIL-2R and CA125, but not neopterin, compared to patients with early-stage disease. However, neopterin was the antigen most often raised in early disease. As for advanced ovarian cancer, preoperative serum sIL-2R levels were higher in patients who developed progressive disease than in those who were progression-free (P = 0.02) after a median follow-up time of 18 months. Furthermore, a trend to higher preoperative serum neopterin values was found in the former patients (P = 0.08). Tumor progression occurred in 3 of 8 (37.5%) patients with low serum preoperative neopterin (< 7.9 nmole/liter) and in 16 of 19 (84.2%) patients with elevated serum neopterin, respectively (P = 0.027). Multivariate analysis on a larger number of patients followed for a longer time is warranted to elucidate the prognostic relevance of these immunologic markers in ovarian cancer. Changes in serum neopterin, sIL-2R, and CA125 levels correlated with the disease course in 50.0, 54.8, and 92.9% of 42 instances, respectively. Moreover, serum CA125 was more sensitive than the other two antigens in the early detection of tumor progression. Therefore serial neopterin and sIL-2R measurements seem to be of limited value in monitoring the disease course in patients with ovarian cancer. PMID:8157196

  4. The activation of polymorphonuclear neutrophils and the complement system during immunotherapy with recombinant interleukin-2.

    PubMed Central

    Baars, J. W.; Hack, C. E.; Wagstaff, J.; Eerenberg-Belmer, A. J.; Wolbink, G. J.; Thijs, L. G.; Strack van Schijndel, R. J.; van der Vall, H. L.; Pinedo, H. M.

    1992-01-01

    The toxicity due to interleukin-2 (IL-2) strongly resembles the clinical picture seen during septic shock. In septic shock activation of polymorphonuclear neutrophils (PMN) and the complement system contribute significantly to the pathophysiology of the condition. We therefore investigated whether similar events contributed to the toxicity observed with IL-2. Four patients received seven cycles of escalating dose IL-2 (18.0 to 72.0 X 10(6) IU m-2 day-1) and 16 were treated with 20 cycles of fixed dose IL-2 (12.0 or 18.0 X 10(6) IU m-2 day-1). Toxicity, as judged by hypotension (P = less than 0.005) and capillary leakage (fall in serum albumin 18.2 vs 4.0 gm l-1; P = less than 0.0005 and weight gain 4.0 vs 1.2 kg; P = less than 0.025) were worse with the esc. dose protocol. PMN became activated following IL-2 with mean peak elastase/alpha 1-antitrypsin (E alpha 1 A) and lactoferrin values of 212 (SEM = 37) and 534 (SEM = 92) ng ml-1 respectively occurring 6 h after the IL-2. Peak values for the esc. dose IL-2 group being generally higher than 500 ng ml-1. Activation of the complement cascade was evidenced by a dose dependent elevation of peak C3a values (fixed dose 9.1 (SEM = 0.6); esc. dose 25.7 (SEM = 6.33); P = less than 0.005) on day 5 of IL-2. There was a significant correlation between C3a levels and the degree of hypotention during the first 24 h after IL-2 (r = 0.91) and parameters of capillary leakage such as weight gain and fall in serum albumin (r = 0.71). These data suggest that activation of PMN initiates endothelial cell damage which subsequently leads to activation of the complement cascade. This latter system then contributes to the haemodynamic changes and capillary leakage seen in IL-2 treated patients. PMID:1733448

  5. Characteristics of murine non-specific killer cells induced in vivo by recombinant human interleukin-2.

    PubMed Central

    Hinuma, S; Naruo, K; Shiho, O; Tsukamoto, K

    1986-01-01

    We have examined the induction of murine non-specific killer cells in vivo and in vitro by purified recombinant human interleukin-2 (rIL-2), and compared their characteristics with respect to killing ability, cell surface phenotypes, and antibody-dependent cell-mediated cytotoxicity (ADCC). C57BL/6 spleen cells cultured with rIL-2 were remarkably cytotoxic against a variety of tumour cells in a 4-hr 51Cr-release assay. Treatment with various antibodies (anti-Thy 1, anti-Lyt 1, anti-Lyt 2, and anti-asialo GM1) plus complement (C) showed that anti-Thy 1 or anti-asialo GM1 antibody plus C removed a majority of killer activity (80% and 66%, respectively). In addition, an increase in ADCC was detected in the spleen cells cultured with rIL-2. These ADCC effector cells were indistinguishable from non-specific killer cells by the cell surface phenotypes. A single administration of rIL-2 in vivo induced only transient and marginal enhancement of non-specific killer activity of spleen cells in C57BL/6 mice. On the other hand, when 10 micrograms of rIL-2 were administered daily by bolus to C57BL/6 mice, the activity increased gradually for about 10 days and reached a plateau. This enhanced non-specific killer activity rapidly decreased and returned to normal by 72 hr after the administration was stopped. The non-specific killer cells induced in vivo in this manner were not only greatly cytotoxic against natural killer (NK)-sensitive tumour cells but were also significantly cytotoxic against NK-resistant tumour cells. Most of the killer activity (more than 90%) was specifically removed by treatment with anti-Thy 1 or anti-asialo GM1 antibody plus C. An increase in ADCC was detected concurrently with an increase in non-specific killer activity in vivo, and both effector cells were indistinguishable by their cell surface phenotypes. These results indicate that a majority of non-specific killer cells induced both in vivo and in vitro by rIL-2 have some common features. Our results also suggest that these cells belong to the same lineage as NK cells, although they are thought to be at different stages from resident NK cells. PMID:3490435

  6. Hypothalamic-pituitary-adrenal activity during chronic central administration of interleukin-2.

    PubMed

    Hanisch, U K; Rowe, W; Sharma, S; Meaney, M J; Quirion, R

    1994-12-01

    The cytokine interleukin-2 (IL-2) exerts numerous effects within the immune as well as the central nervous system and is thought to serve as a humoral signal in their communication. Brain-derived or blood-borne IL-2 may also control the activity of the hypothalamic-pituitary-adrenal (HPA) axis at various levels of regulation. In this study we investigated whether persistently elevated levels of central IL-2, which are associated with several diseases or induced during immunotherapeutic use of this cytokine, could induce long term activation of the HPA axis. Adult male Sprague-Dawley rats received an intracerebroventricular infusion of the recombinant cytokine at a rate of 5 U/h (equivalent to 2.5 ng/h or 162 fmol/h) by means of osmotic minipumps. Control animals received heat-inactivated IL-2. After 7 days of continuous infusion, blood samples were taken at intervals of 4 h over a period of 24 h, and plasma levels of ACTH and corticosterone (CORT) were determined. IL-2 caused a significant increase in ACTH levels during the later portion of the dark phase of the cycle. Plasma CORT concentrations were significantly elevated over almost the whole diurnal cycle. Measurements of CORT-binding globulin concentrations revealed IL-2-induced decreases during the dark phase, resulting in a marked increase in free CORT. Additionally, after 11 days of chronic infusion, both groups of animals underwent a 20-min restraint stress. IL-2-treated animals showed stress-induced increases in plasma ACTH and CORT that were not significantly different from those of animals treated with heat-inactivated IL-2. Along with the alteration of HPA activity seen in the IL-2-treated animals, chronic delivery of the cytokine caused periventricular tissue damage and gliosis. Taken together, the data reflect the capacity of IL-2 to modulate neuroendocrine activity over an extended period of treatment. Moreover, the IL-2-induced effects on HPA activity seen here may help to explain some of the endocrine disturbances seen in patients undergoing IL-2 immunotherapy. PMID:7988433

  7. Regulation of interleukin-2 signaling by fatty acids in human lymphocytes.

    PubMed

    Gorjão, Renata; Hirabara, Sandro Massao; de Lima, Thaís Martins; Cury-Boaventura, Maria Fernanda; Curi, Rui

    2007-09-01

    Docosahexaenoic (DHA; C22:6 n-3), eicosapentaenoic (EPA; C20:5 n-3), palmitic (PA; C16:0), and stearic (SA; C18:0) acids decrease lymphocyte proliferation in concentrations of >50 muM, as observed in our previous study. However, oleic acid (OA; C18:1 n-9) and linoleic acid (LA; C18:2 n-6) increase lymphocyte proliferation at 25 muM. In this study, the effect of these FAs on the interleukin-2 (IL-2) signaling pathway in human lymphocytes was investigated. Cells were isolated from heparinized venous blood of healthy human donors by density-gradient sedimentation. Cells were stimulated with 5 mug/ml concanavalin A and treated with FAs in the absence or presence of IL-2 for 1 hour. CD25-alpha externalization was analyzed by flow cytometry, and Janus kinase 1 (JAK1), JAK3, signal transducer and activator of transcription (STAT) 5, extracellular signal-regulated kinases (ERKs) 1 and 2, Akt, and protein kinase C (PKC)-zeta phosphorylation were analyzed by Western blotting. The expression of CD25-alpha at the cell surface was increased by DHA, SA, and PA but was unaffected by EPA, OA, and LA. PA, SA, DHA, and EPA decreased JAK1, JAK3, STAT5, and Akt phosphorylation induced by IL-2, but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation, whereas the other FAs caused a marked decrease. PKC-zeta phosphorylation was decreased by OA and LA and was not altered by the remaining FAs. In conclusion, the inhibitory effect of PA, SA, DHA, and EPA on lymphocyte proliferation observed in our previous study was attributable to a decrease in JAK/STAT, ERK, and Akt pathways activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK1/2 phosphorylation through PKC-zeta activation. The inhibition of JAK1, JAK3, STAT5, ERK1/2, and Akt phosphorylation caused by DHA, SA, and PA is associated with an alteration of CD25 expression at the cell surface. PMID:17592174

  8. Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals.

    PubMed Central

    Oyaizu, N; Chirmule, N; Pahwa, S

    1992-01-01

    The CD4 molecule plays an essential role in antigen-induced activation of T helper (Th) cells, but its contribution to signal transduction events resulting in physiologic T cell function is ill defined. By utilizing anti-CD4 monoclonal antibodies (MAbs) that recognize distinct epitopes of CD4, we have investigated the role of CD4 molecule in antigen-induced interleukin 2 (IL-2) and IL-2 receptor (IL-2R) alpha chain expression in class II major histocompatibility complex-restricted antigen-specific human Th clones. Pretreatment of the Th clones with Leu3a resulted in a dose-dependent suppression of antigen-induced proliferative responses, inositol phosphate accumulation, increase in free cytoplasmic calcium ions ([Ca2+]i), IL-2 mRNA accumulation, IL-2 secretion, and membrane IL-2R expression. IL-2R mRNA accumulation, however, was unaffected even at highest Leu3a concentrations. Leu3a treatment did not affect bypass activation of T cells with PMA plus ionomycin or activation via CD2 molecule. The MAb OKT4, which binds another domain of CD4, was not inhibitory. These results suggest that after T cell antigen receptor-CD3 activation, IL-2 gene induction, IL-2 secretion, and membrane IL-2R expression are absolutely dependent upon participation of CD4 molecules, phosphatidylinositol (PI) hydrolysis, and increase in [Ca2+]i. The requirement for IL-2R gene induction, however, occurs independently of CD4 molecule participation and PI hydrolysis. Images PMID:1534818

  9. Interleukin-1 alpha, soluble interleukin-2 receptor, and IgG concentrations in cystic fibrosis treated with prednisolone.

    PubMed Central

    Greally, P; Hussain, M J; Vergani, D; Price, J F

    1994-01-01

    The cytokines interleukin-1 and interleukin-2 participate in the inflammatory response, and may contribute to hypergammaglobulinaemia G and the development of lung injury in cystic fibrosis. Anti-inflammatory treatment with corticosteroids may attenuate this response. The effect of a 12 week course of oral prednisolone on spirometry and serum concentrations of interleukin-1 alpha (IL-1 alpha), soluble interleukin-2 receptor (sIL-2R), and IgG was investigated in 24 children with cystic fibrosis. Prednisolone was administered, in a double blind and placebo controlled manner, at an initial dose of 2 mg/kg daily for 14 days and tapered to 1 mg/kg on alternate days for 10 weeks. The treated group (n = 12) experienced an increase in forced expiratory volume in one second and forced vital capacity at 14 days, however, these changes were smaller at 12 weeks. In the treated group, change in pulmonary function was associated with decreased serum IgG and cytokine concentrations. Prednisolone suppresses serum concentrations of these cytokines, which may participate in the inflammatory response, the excessive synthesis of IgG, and airflow obstruction observed in cystic fibrosis patients. PMID:8067791

  10. Cyclosporine inhibits expression of receptors for interleukin 2 and transferrin on mitogen-activated human T lymphocytes

    SciTech Connect

    John, J.K.; Prince, H.E.

    1986-03-05

    Cyclosporine (CyS) has been shown to inhibit activation of human T lymphocytes. A cascade of interactions involving Interleukin 2 (IL2), Interleukin 2 receptor (IL2R) and transferrin receptor (TR) is necessary for activation. In studies using peripheral blood mononuclear cells obtained from healthy donors, the authors measured the expression of IL2 and TR (using receptor-specific monoclonal antibodies and flow cytometry) and DNA synthesis (/sup 3/-thymidine incorporation) in response to PHA, OKT3, Leu 4 or Con A. In the presence of CyS (0.5 ..mu..g/ml) expression of IL2R and TR as well as DNA synthesis were markedly reduced. Upon serial dilutions of CyS, changes in DNA synthesis in response to OKT3 reflected changes in IL2R and TR levels, indicating all 3 parameters may be interrelated. Addition of exogenous IL2 partially abrogated the inhibitory effect of CyS on these activation parameters in response to PHA, OKT3 and Leu 4 but not to Con A. The authors results suggest that CyS is an effective inhibitor of mitogen-induced expression of IL2R and TR and that exogenous IL2 partially reverses this inhibitory effect.

  11. NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

    SciTech Connect

    Zhao Guohua; Shi Lingfang; Qiu Daoming; Hu Hong; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2005-05-01

    NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

  12. Wallenda regulates JNK-mediated cell death in Drosophila

    PubMed Central

    Ma, X; Xu, W; Zhang, D; Yang, Y; Li, W; Xue, L

    2015-01-01

    The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. Here, we show that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, we find that Wnd triggers JNK activation and cell death via its kinase domain. Moreover, we show that both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of scribble-induced JNK-dependent cell death. Thus, our data suggest that wnd encodes a novel essential cell death regulator in Drosophila. PMID:25950467

  13. Inhibitory effect of 3'-azido-3'-deoxythymidine on proliferative responsiveness of CD8+ lymphocytes to interleukin-2.

    PubMed

    Mercure, L; Lalonde, R; Phaneuf, D; Brenner, B; Wainberg, M A

    1994-07-01

    Although several studies have shown that 3'-azido-3'-deoxythymidine (AZT) is not toxic for CD4+ lymphocytes, its effect on CD8+ cells has never been studied in a systematic way. We purified CD8+ cells from the peripheral blood mononuclear cells of both human immunodeficiency virus (HIV)-seronegative and HIV-infected individuals by means of magnetic beads that had been coated with monoclonal antibodies. We report that AZT, but not two other nucleosides tested, inhibited the interleukin-2-dependent proliferation of CD8+ lymphocytes in a dose-dependent manner. No such effect was observed with regard to CD4(+)-enriched populations. The AZT-mediated antiproliferative effect did not appear to be related to either the CD4+ count or to prior treatment with this drug in the case of HIV-seropositive subjects. PMID:8556489

  14. Phytohemagglutinin induced proliferation by aged lymphocytes: reduced expression of high affinity interluekin-2 receptors and interleukin-2 secretion

    SciTech Connect

    Froelich, C.J.; Burkett, J.S.; Guiffaut, S.; Kingsland, R.; Brauner, D.

    1988-01-01

    Human lymphocytes from elderly and young donors were cultured with phytohemagglutinin. Cultures from two groups of aged donors, recruited respectively from the authors ambulatory clinic and a nursing home, incorporated less tritiated thymidine (/sup 3/H-TdR) and secreted less interleukin-2 than did young donors. Furthermore, as determined for the first time by a radioligand binding receptor assay, the aged lymphoblasts possessed significantly fewer high affinity IL-2 receptors per cell. Despite a decrease in the number of high affinity receptor cells the dissociation constant (Kd) was comparable for the three groups. It was also shown that the amounts of soluble IL-2 receptors that were released into the supernatants by mitogen stimulated cells did not differ for the aged and young donors. These data suggest that defects in IL-2 production and high affinity IL-2 receptor generation may both be responsible for immune deficiency in the elderly.

  15. Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in a murine breast cancer model.

    PubMed Central

    Addison, C L; Braciak, T; Ralston, R; Muller, W J; Gauldie, J; Graham, F L

    1995-01-01

    Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers. PMID:7667323

  16. Intratumoral Injection of an Adenovirus Expressing Interleukin 2 Induces Regression and Immunity in a Murine Breast Cancer Model

    NASA Astrophysics Data System (ADS)

    Addison, Christina L.; Braciak, Todd; Ralston, Robert; Muller, William J.; Gauldie, Jack; Graham, Frank L.

    1995-08-01

    Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

  17. Formation of an active form of the interleukin-2/15 receptor beta-chain by insertion of the intracisternal A particle in a radiation-induced mouse thymic lymphoma and its role in tumorigenesis.

    PubMed

    Ukai, Hideki; Ishii-Oba, Hiroko; Ukai-Tadenuma, Maki; Ogiu, Toshiaki; Tsuji, Hideo

    2003-06-01

    Although many reports suggest that aberrant regulation of cytokine signaling pathways via the interleukin-2 receptor (IL-2R) induces tumorigenic transformation, constitutively active IL-2R in tumors has not been reported. We searched for genomic alteration of the IL-2/15R beta-subunit gene (IL-2/15R beta) in cytokine-independent cell lines established from radiation-induced mouse thymic lymphomas. In the TL34 cell line and its primary tumor, one of the IL-2/15R beta alleles was rearranged by the insertion of an intracisternal A particle (IAP) retrotransposon. The IAP-IL2/15R beta chimeric gene expressed chimeric mRNA in which IAP-coding Gag-Pol mRNA was fused to IL-2/15R beta mRNA and coded for Gag-Pol-IL-2/15R beta chimeric protein. Forced expression of the Gag-Pol-IL-2/15R beta chimeric cDNA in a mouse cytotoxic T-cell line (CTLL-2) converted IL-2-dependent cell growth to IL-2-independent growth, suggesting that the chimeric protein activates some of the IL-2 signaling pathways necessary for cell proliferation. Downregulation of the expression of the Gag-Pol-IL-2/15R beta chimeric protein in TL34 by antisense RNA inhibited cell growth, and concomitantly reduced the level of c-myc protein. These results suggest that the Gag-Pol-IL-2/15R beta is a constitutively active form that transmits proliferative signals by expressing downstream target genes, including c-myc. Thus, we demonstrated that the chimeric receptor gene produced by the insertion of an IAP functions as an oncogene by providing IL-2-independent autonomous growth potential. PMID:12766910

  18. Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens by activating natural killer cells (NK), cytotoxic T lymphocytes, and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such i...

  19. Human monoclonal natural autoantibodies against the T-cell receptor inhibit interleukin-2 production in murine T cells

    PubMed Central

    Robey, Ian F; Schluter, Samuel F; Akporiaye, Emmanuel; Yocum, David E; Marchalonis, John J

    2002-01-01

    Natural autoantibodies (NAAbs) specific for the T-cell receptor (TCR) are present in all human sera, but individuals with rheumatoid arthritis (RA) generally produce higher titres of immunoglobulin M (IgM) isotype autoantibodies (AAbs) against V? TCR epitopes. To investigate possible correlations between the specificity of such AAbs and their role in immunomodulation, we generated seven B-cell hetero-hybridomas, secreting monoclonal IgM NAAbs, from the synovial tissue and peripheral blood of patients with RA. Here we report three anti-TCR monoclonal autoantibodies (mAAbs) OR2, OR5 and Syn 2H-11 with the ability to bind subsets of murine T cells, including the ovalbumin-specific DO-11.10 clone. These antibodies did not induce apoptosis in vitro, but prevented interleukin-2 (IL-2) production by antigen-specific T cells. These findings suggest an immunomodulatory function for NAAbs to TCR V-region epitopes and serve as the foundation for testing human anti-TCR mAAbs in animal models with the eventual goal of using them as therapeutic agents in human disease. PMID:11985662

  20. Respective contribution of intracellular calcium release and extracellular calcium influx for interleukin-2 synthesis in activated T-cell hybrids.

    PubMed Central

    Williams, D B; Perera, M A; Dorrington, K J; Klein, M H

    1990-01-01

    Triggering of the T-cell receptor (TcR)alpha beta/CD3 receptor complex with anti-allotypic antibodies or concanavalin A (Con A) induced a rapid release of intracellular calcium in a murine T-cell hybridoma model system. Internal calcium release preceded the influx of extracellular calcium, as judged by comparative analysis of time-dependent changes in Quin 2 fluorescence following T-cell activation in the presence and absence of extracellular calcium. The magnitude of intracellular calcium release and extracellular calcium influx depended on the degree of receptor-occupancy and cross-linking. Correlations between the concentration of stimulating ligand, cytosolic calcium increase and IL-2 synthesis indicated a positive but non-linear relationship. Our data suggest that TcR cross-linking may provide a third T-cell activation signal which, in conjunction with protein kinase C activation and cytosolic calcium elevation, together form a signal triad responsible for interleukin-2 (IL-2) synthesis. PMID:2312169

  1. Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production

    PubMed Central

    Zeiser, Robert; Nguyen, Vu H.; Beilhack, Andreas; Buess, Martin; Schulz, Stephan; Baker, Jeanette; Contag, Christopher H.; Negrin, Robert S.

    2006-01-01

    CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect. (Blood. 2006;108:390-399) PMID:16522809

  2. In vitro assessment of choline dihydrogen phosphate (CDHP) as a vehicle for recombinant human interleukin-2 (rhIL-2)

    PubMed Central

    Foureau, David M.; Vrikkis, Regina M.; Jones, Chase P.; Weaver, Katherine D.; MacFarlane, Douglas R.; Salo, Jonathan C.; McKillop, Iain H.; Elliott, Gloria D.

    2013-01-01

    Choline dihydrogen phosphate (CDHP) is an ionic liquid reported to increase thermal stability of model proteins. The current work investigated CDHP effect on structural integrity and biological activity of recombinant human interleukin-2 (rhIL-2), a therapeutic protein used for treating advanced melanoma. In vitro CDHP biocompatibility was also evaluated using primary cell cultures, or B16-F10 cell line, chronically exposed to the ionic liquid. Formulation of rhIL-2 in an aqueous 680mM CDHP pH 7.4 solution resulted in a 12.5C increase in the Tm of rhIL-2 compared to a basic buffer formulation, and provided conformational rhIL-2 stabilization when the solution was heated to 23.3C above the Tm. CDHP solutions (?80mM), exhibited no cytotoxic activity toward primary splenocytes or B16-F10 cells in culture. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30mM CDHP aqueous solution with NaHCO3 (pH?7.2) compared to controls without CDHP. While increased Tm is associated with a diminished rhIL-2 biological activity, the therapeutic protein remains structurally intact and functional. PMID:24504148

  3. Antiapoptotic effect of interleukin-2 (IL-2) in B-CLL cells with low and high affinity IL-2 receptors.

    PubMed

    Decker, Thomas; Bogner, Christian; Oelsner, Madlen; Peschel, Christian; Ringshausen, Ingo

    2010-11-01

    Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins. PMID:20544350

  4. Oral tacrolimus oil formulations for enhanced lymphatic delivery and efficient inhibition of T-cell's interleukin-2 production.

    PubMed

    Yoshida, Takayuki; Nakanishi, Kiyo; Yoshioka, Tatsunobu; Tsutsui, Yuuki; Maeda, Atsushi; Kondo, Hiromu; Sako, Kazuhiro

    2016-03-01

    Oral oil formulations have been reported to deliver drugs into the lymph. Lymphatic delivery of immunomodulatory drugs can more efficiently expose the drugs to T-cells in lymph, consequently induce higher efficacy and lower side effects. In this study, effects of tacrolimus oral oil formulations on drug blood exposure, and on inhibition of T-cell's interleukin-2 (IL-2) production were investigated in rats. Oil formulations (sunflower oil, cacao butter, medium chain triglyceride, and palm oil) dissolving tacrolimus showed lower drug blood concentration than a solid dispersion formulation (SDF). The sunflower oil, and cacao butter formulations suppressed drug blood exposure to 50% of the SDF, and inhibited T-cell's IL-2 production similar to the SDF. In vitro digestion tests indicated that slower digestion of the oils might reduce amount and rate of tacrolimus blood absorption. The cacao butter formulations showed 3.0 times more rapid tacrolimus absorption to lymphatic fluid than the SDF. Ratio of the rate constants of absorption into lymph to that into blood was higher in oil formulations (15 times in cacao butter, 15 times sunflower oil, and 3.5 times palm oil) than in the SDF. These results indicated that the oral oil formulations might be suitable for reduced tacrolimus blood concentration for low systemic side effects, and keep high lymph concentration for high efficacy in organ transplantation patients. PMID:26748381

  5. Characterization of a human X mouse T cell hybridoma and identification of a clone secreting and binding interleukin-2.

    PubMed Central

    Durrant, L G; Parkar, M; Kenworthy, N; Taylor, G M

    1984-01-01

    Human lymphocytes stimulated with phytohaemagglutinin (PHA) were fused with an HGPRT- murine lymphoma, BW5147, and a hybridoma BwFc93-1 was isolated and cloned in agarose. This human X mouse hybrid and nine clones derived from it were characterized by chromosome analysis, phenotypic and functional assays. Karyotyping and isoenzyme studies showed the presence of five human chromosomes in BwFc93-1 with preferential retention of three chromosomes--6, X and 15--in the clones. Membrane immunofluorescence analysis revealed that all the clones expressed human and mouse class 1 MHC antigens and the mouse T cell antigens Thy-1 and T200, but were devoid of human OKT3, OKT8 and mouse Lyt-2. Human OKT4 and OKM1 phenotypes were transiently expressed by one clone and mouse Lyt 1 by two other clones. Several T4-, Lyt-1- clones produced and bound human interleukin-2 (IL-2) indicating a lack of correlation between human T cell phenotype and function in those hybrids. There was also evidence of dichotomy in the secretion of IL-2 and expression of the IL-2 receptor since clones were identified which either bound or secreted IL-2. One clone expressing IL-2 receptors could be induced to produce human IL-2 by simultaneously stimulating with PHA and phorbol myristate acetate (PMA). Images Figure 1 Figure 2 PMID:6609121

  6. Treatment of metastatic renal cell carcinoma with subcutaneous interleukin 2: evidence for non-renal clearance of cytokines.

    PubMed Central

    Banks, R. E.; Forbes, M. A.; Hallam, S.; Jenkins, A.; Wadhwa, M.; Dilger, P.; Meager, A.; Thorpe, R.; Bowmer, C. J.; Joffe, J. K.; Patel, P.; Johnson, P. W.; Selby, P. J.

    1997-01-01

    The circulating cytokine concentrations following administration of subcutaneous recombinant interleukin 2 (IL-2) in combination with interferon alpha and 5-fluorouracil used to treat advanced renal cancer were studied. One patient was anephric and on dialysis, and seven had normal biochemical renal function, although five had undergone single nephrectomy. The pharmacokinetics of IL-2 and changes in IL-6 and tumour necrosis factor (TNF)-alpha were essentially similar in all patients including the anephric patient, irrespective of the periods of dialysis, although at some time points, IL-2 concentrations were slightly higher in the anephric patient than in the others. These results show that for subcutaneous administration of low-dose IL-2, renal clearance of IL-2 is not important. This contrasts with high-dose, intravenous IL-2 where blood concentrations are higher and renal clearance seems to occur, perhaps because of saturation of the non-renal mechanisms of clearance. The subcutaneous route is certainly preferred if IL-2 is used in anephric patients and in those with impaired renal function, and it may be generally preferred for most purposes. PMID:9192992

  7. Treatment of Walker ascites tumor cells by combination of photodynamic therapy with cyclophosphamide and interleukin-2 entrapped in liposomes

    NASA Astrophysics Data System (ADS)

    Dima, Vasile F.; Ionescu, Mircea D.; Balotescu, Carmen; Dima, V. S.

    2003-12-01

    The purpose of this study was to investigate the beneficial and adverse local effects of PDT associated with chemoimmunotherapy on rats bearing Walker ascites tumor cells. Experiments were performed on five batches of Wistar inbred rats with ascites tumor cells receiving intraperitoneally PDT (Photofrin II and 18 hrs later HeNe laser irradiation); Cyclophosphamide (CY); interleukin-2 (IL-2) or associated therapy (PDT+CY+IL-2). The control batch consisted of untreated rats (HBSS). The following results were noticed: (a) sole administration of PDT, IL-2 or CY reduced tumor growth, gave survival rates between 28.4 and 56.5% and cure rates ranging from 12.4 to 33.3%; (b) combined therapy (PDT+CY+IL-2) decreased tumor growth, increased survival rates (88.5%) and cure rates were 73.1% forty-two days post-transplantation. Summing up, in this study we noticed that PDT associated with chemoimmunotherapy reduced mortality as well as tumor volumes and increased cure rates in rats with ascites tumor cells. This approach points to the need for further evaluation in patients with peritoneal malignancies.

  8. Influence of tunicamycin, sialidase, and cholera toxin on gangliosides and T-lymphocyte responses to interleukin 2

    SciTech Connect

    Semmes, O.J.; Bailey, J.M.; Merritt, W.D.

    1986-05-01

    The authors have shown that gangliosides inhibit interleukin 2 (IL 2)-dependent proliferation of murine T cells. Tunicamycin (TM), sialidase, and cholera toxin-..beta.. subunit (..beta..-CT) are known modulators of cell surface glycoconjugates. To test the possible role of endogenous gangliosides in T cell responses to IL-2, the effect of these agents on ganglioside expression and cell proliferation was studied. Gangliosides were labelled for 24 hrs with /sup 3/H-glucosamine/galactose in the presence of IL-2 and purified sialidase, TM or ..beta..-CT. Gangliosides were isolated and the species separated by TLC. Alternatively, proliferation was assayed by /sup 3/H-thymidine uptake after 48 hrs culture. TM treatment at a concentration (10 ..mu..g/ml) that completely inhibited proliferation resulted in a 86% reduction of incorporation of saccharide precursors into gangliosides compared to a 50% reduction into proteins. Sialidase treatment (0.1 IU/ml) resulted in a 70% inhibition of proliferation and 30% reduction of radiolabel into gangliosides, of which 3 species were specifically reduced. ..beta..-CT, which binds to GM/sub 1/ and to a lesser extent GD/sub 1a/, caused a 50% reduction in proliferation response at 35 units/ml. The results support the hypothesis that gangliosides are involved in IL-2-dependent proliferation.

  9. The association of -330 interleukin-2 gene polymorphism with its plasma concentration in Iranian multiple sclerosis patients.

    PubMed

    Sayad, Arezou; Movafagh, Abolfazl

    2014-01-01

    Multiple sclerosis (MS) is a chronic neuroinflammatory demyelinating disease of the central nervous system. The cytokine genes are involved in autoimmune diseases such as MS. In this study, we report the influence of -330 interleukin-2 (IL2) gene polymorphism on its plasma levels in a group of Iranian MS patients. In this study 100 MS patients and 100 ethnically, age, and sex matched healthy controls were selected from Medical Genetics Department of Sarem Women Hospital. Blood samples of all individuals were collected in EDTA tubes. The restriction fragment length polymorphism PCR (RFLP) method was applied to determine various alleles and genotypes in these individuals. Plasma concentration of IL2 was measured in all the samples using human IL2 kit. The frequency of -330 T/T IL2 genotype was higher in MS patients compared to normal individuals. Accordingly, the plasma levels of IL2 were significantly higher (P < 0.0001) in patients when compared to the control group. In conclusion, in case of MS patients the -330 T/T IL2 genotype is associated with higher plasma levels of IL2. PMID:24959373

  10. Influence of immunomodulatory drugs on the cytotoxicity induced by monoclonal antibody 17-1A and interleukin-2.

    PubMed

    Flieger, Dimitri; Varvenne, Michael; Kleinschmidt, Rolf; Schmidt-Wolf, Ingo G H

    2007-03-01

    Patients treated with monoclonal antibodies and cytokines for cancer receive often co-medication, which may influence treatment efficacy. Therefore, we investigated with a flowcytometric cytotoxicity assay the effect of several immunomodulatory drugs on antibody dependent cellular cytotoxicity (ADCC), interleukin-2 (IL-2) induced cytotoxicity and IL-2-induced-ADCC. We found that dexamethasone markedly inhibited the IL-2 induced cytotoxicity and the IL-2-induced-ADCC. Ondansetron, a 5-HT-3 serotonin receptor antagonist augmented significantly ADCC. Clemastine, a histamine type-2 receptor antagonist augmented the IL-2-induced-ADCC. The TNF antagonist thalidomide suppressed ADCC whereas pentoxifylline proved to be ineffective. Other tested drugs namely ibuprofen and indomethacin, both prostaglandin E2 antagonists, cimetidine a histamine type-2 receptor antagonist, the opioid pethidine, prostaglandin E2 and histamine exerted minor effects or had no influence on the tested parameters. We conclude that glucocorticosteroids should be avoided with monoclonal antibody and cytokine treatment. According to our in vitro data the other drugs tested did not have a negative impact on cellular cytotoxicity and ADCC. PMID:17562330

  11. Keratinocyte-derived T-cell growth factor: a T-cell growth factor functionally distinct from interleukin 2.

    PubMed Central

    Kupper, T S; Coleman, D L; McGuire, J; Goldminz, D; Horowitz, M C

    1986-01-01

    T-cell growth factor, more recently termed interleukin 2 (IL-2), is the product of activated T lymphocytes and is considered the principal trophic factor for T lymphocytes. The activity of IL-2 preparations is assessed by the degree to which they support the growth of various IL-2-dependent cell lines. We report that murine epidermal epithelial cells (keratinocytes) produce and release a factor that supports the growth of the helper-T-cell-derived, IL-2-dependent cell line HT-2. This substance, keratinocyte-derived T-cell growth factor (KTGF), does not support the growth of an IL-2-dependent cell line derived from cytotoxic T cells (line CTLL-2). This differential effect on IL-2-dependent cell lines is unique to KTGF. KTGF has an apparent molecular weight of 25,000-35,000 and has properties similar to those of conventional IL-2 by reversed-phase and gel-filtration HPLC analysis. However, even highly purified KTGF fails to stimulate the proliferation of CTLL-2 cells. The observation that epidermal epithelium produces a trophic factor for T lymphocytes may help explain the basis for preferential proliferation of T cells in the microenvironment of skin in certain dermatologic disorders. Further, it suggests that different IL-2-dependent T-cell lines may have distinct growth requirements and that non-lymphocyte cell types may produce factors capable of maintaining the growth of T cells. PMID:3520573

  12. Modulating effect of interleukin 2 therapy on interferon production by blood leukocytes of patients with minimal residual hematological disease.

    PubMed

    Kandefer-Szerszeń, M; Legieć, W; Dmoszyńska, A; Szuster-Ciesielska, A

    1997-01-01

    This study was designed to investigate the effect of 1.8 x 10(6) U/day interleukin 2 (IL-2) therapy on interferon (IFN) production. Patients enrolled in the study suffered from multiple myeloma (MM), Hodgkin's disease (HD) and non-Hodgkin lymphoma (NHL). All of them were in remission after chemotherapy or radiotherapy. Results indicated that IL-2 given subcutaneously at a dose of 1.8 x 10(6) U/day for 3 weeks induced IFN-gamma in serum of patients and caused a prolonged effect on the ability of blood leukocytes to produce IFN-gamma after stimulation in vitro by mitogen phytohemagglutinin (PHA). Such enhancement of IFN-gamma production may be beneficial for antitumor immune response. Low-dose IL-2 therapy was well tolerated by all patients and side effects not exceeding II grade of toxicity according to WHO scale were observed. Five patients with MM have relapsed 3-10 months after cesation of IL-2 therapy but 15 patients 18 months after therapy were in complete remission. PMID:9597084

  13. Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes.

    PubMed Central

    Gilmour, K C; Pine, R; Reich, N C

    1995-01-01

    Although prolactin and interleukin 2 (IL-2) can elicit distinct physiological responses, we have found that their signal pathways share a common signal transducer and activator of transcription, STAT5. STAT5 was originally identified as a mammary gland factor induced by prolactin in lactating breast cells. Here we demonstrate that STAT5 is activated after IL-2 stimulation of two responsive lymphocyte cell lines, Nb2 and YT. Activation of STAT5 is measured both by IL-2-induced tyrosine phosphorylation and by IL-2-induced DNA binding. The STAT5 DNA recognition site is the same as the interferon gamma-activated site (GAS) in the interferon regulatory factor 1 gene. We demonstrate that the GAS element is necessary and sufficient for transcriptional induction by both IL-2 and prolactin in T lymphocytes. These results indicate that the role of STAT5 in the regulation of gene expression is not restricted to mammary cells or to prolactin, but is an integral part of the signal pathway of a critical immunomodulatory cytokine, IL-2. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479881

  14. Normal gamma interferon (IFN-. gamma. ) and decreased interleukin-2 (IL-2) production by copper-deficient mice

    SciTech Connect

    Lukasewycz, O.A.; Prohaska, J.R. )

    1991-03-15

    The production of both interleukin-2 (IL-2) and gamma interferon (IFN-{gamma}) was determined in lymphocyte preparations from spleens of copper-deficient ({minus}Cu) and copper adequate control (+Cu) mice. Swiss albino mice were fed a diet low in copper. The +Cu mice drank water with copper added, while {minus}Cu mice drank deionized water. Compared to +Cu controls, {minus}Cu mice had lower hematocrits, reduced levels of liver Cu, low plasma ceruloplasmin activity, and higher levels of liver iron. Production of IL-2 was assessed by the response of an IL-2-dependent cell line (CTLL) to serial dilutions of Con A-stimulated splenic lymphocyte culture supernatants. IFN-{gamma} levels were determined in these same supernatants by an enzyme-linked immunosorbant assay. Analysis indicated that IL-2 production by splenic lymphocytes from {minus}Cu mice was only 62% of the mean +Cu value. IFN-{gamma} levels of {minus}Cu and +Cu splenic lymphocytes, on the other hand, were equivalent. These data indicate differential effects of copper deficiency on two distinct lymphokines elaborated by the same murine T-help subpopulation, T{sub H}1.

  15. The clinical effects of prolonged treatment of patients with advanced cancer with low-dose subcutaneous interleukin-2 [corrected

    PubMed Central

    Stein, R. C.; Malkovska, V.; Morgan, S.; Galazka, A.; Aniszewski, C.; Roy, S. E.; Shearer, R. J.; Marsden, R. A.; Bevan, D.; Gordon-Smith, E. C.

    1991-01-01

    Thirty-five patients with advanced malignant disease have been treated as outpatients with increasing doses (0.1-100 mcg) of interleukin 2 (IL2) by once daily self-administered subcutaneous (s.c.) injection, 5 days weekly for 8 weeks followed by a 4 week observation period. Systemic side effects were not experienced by patients at the 3 lower doses. Three patients required dose reduction from 100 mcg daily because of intolerance (fever, rash, lethargy, nausea and vomiting) and one patient was discontinued because of dyspnoea. We observed immunological effects at the 100 mcg dose (but not at the lower doses). These consisted of (a) a modest sustained lymphocytosis, (b) eosinophilia in six (out of nine) patients and (c) a significant rise in IL2-stimulated peripheral blood lymphocyte activated killer (LAK) cell activity in six (out of nine) patients to a mean of 2.0 times pretreatment levels (P less than 0.01). Two (out of nine) patients with renal cell carcinoma treated with 100 mcg daily had partial responses of duration 4 and 9 months respectively and a further three had disease stabilisation for at least 3 months. Low dose long-term s.c. IL2 is clinically and immunologically active, and in comparison to other IL2 regimens it has minor toxicity and is easy to administer. These characteristics make low dose s.c. IL2 suitable for study in the adjuvant setting. PMID:1997106

  16. The clinical effects of prolonged treatment of patients with advanced cancer with low-dose subcutaneous interleukin-2 [corrected].

    PubMed

    Stein, R C; Malkovska, V; Morgan, S; Galazka, A; Aniszewski, C; Roy, S E; Shearer, R J; Marsden, R A; Bevan, D; Gordon-Smith, E C

    1991-02-01

    Thirty-five patients with advanced malignant disease have been treated as outpatients with increasing doses (0.1-100 mcg) of interleukin 2 (IL2) by once daily self-administered subcutaneous (s.c.) injection, 5 days weekly for 8 weeks followed by a 4 week observation period. Systemic side effects were not experienced by patients at the 3 lower doses. Three patients required dose reduction from 100 mcg daily because of intolerance (fever, rash, lethargy, nausea and vomiting) and one patient was discontinued because of dyspnoea. We observed immunological effects at the 100 mcg dose (but not at the lower doses). These consisted of (a) a modest sustained lymphocytosis, (b) eosinophilia in six (out of nine) patients and (c) a significant rise in IL2-stimulated peripheral blood lymphocyte activated killer (LAK) cell activity in six (out of nine) patients to a mean of 2.0 times pretreatment levels (P less than 0.01). Two (out of nine) patients with renal cell carcinoma treated with 100 mcg daily had partial responses of duration 4 and 9 months respectively and a further three had disease stabilisation for at least 3 months. Low dose long-term s.c. IL2 is clinically and immunologically active, and in comparison to other IL2 regimens it has minor toxicity and is easy to administer. These characteristics make low dose s.c. IL2 suitable for study in the adjuvant setting. PMID:1997106

  17. Subcutaneous low-dose recombinant interleukin 2 and alpha-interferon in patients with metastatic renal cell carcinoma.

    PubMed Central

    Ravaud, A.; Négrier, S.; Cany, L.; Merrouche, Y.; Le Guillou, M.; Blay, J. Y.; Clavel, M.; Gaston, R.; Oskam, R.; Philip, T.

    1994-01-01

    A double-institution phase II study was performed in patients with metastatic renal cell carcinoma treated subcutaneously (s.c.) with interleukin 2 (IL-2) and alpha-interferon (INF-alpha). Thirty-eight patients were treated over a course of 7 weeks. Initially (day 1 + 2) patients received s.c. IL-2 at 18 x 10(6) IU m-2. During the following 6 weeks, patients received s.c. IL-2 at 3.6 x 10(6) IU m-2 for 5 days per week and s.c. INF-alpha at 5 x 10(6) for 3 days per week. Thirty-eight patients were evaluated for response. An objective response was seen in seven patients (18.4 +/- 12.3%), with one complete response and six partial responses. Median duration of response was 6.7 months. Toxicity could be evaluated in 38 patients and was limited. Mild to moderate toxicity included fever (97%), fatigue or malaise (76%), nausea or vomiting (50%), anorexia (32%), hypotension (26%), neurological disturbances (26%) and hypercreatininaemia (39%). In addition, four grade IV haematological toxicities were noted. No cardiac side-effects were seen. IL-2 and INF-alpha given by this schedule can be safely administered in an outpatient setting. The objective response rate was similar to our previous treatments with high-dose IL-2 given as a continuous infusion. PMID:8198979

  18. Comparison of the radiosensitivity of interleukin-2 production between species, between tissues, and between young and old individuals

    SciTech Connect

    Peterson, W.J.; Akagawa, T.; Anderson, D.G.; Makinodan, T.

    1985-04-01

    The radiosensitivity of interleukin-2 (IL-2) production was assessed of (a) peripheral blood mononuclear cells (PBMC) of young humans, dogs, and mice (C57BL/6); (b) PBMC and splenic cells of young mice; and (c) PBMC of young and old humans and the splenic cells of young and old mice. The results indicate that (a) large differences in radiosensitivity exist between the PBMC of humans, dogs, and mice (e.g., the radiation doses which resulted in 37% remaining IL-2 activity (D37) of human, dog, and mouse PBMC were 3771, greater than 10,000, and 1398 rads, respectively); (b) only a small difference exists between the PBMC and splenic cells of mice; and (c) no difference exists between the PBMC of young and old humans and between splenic cells of young and old mice. Topological abnormalities, as judged by scanning electron microscopic analysis, could not be detected in dog PBMC after their exposure to 1800 rads, but could be detected in mouse PBMC after their exposure to 400 rads.

  19. Effect of interleukin-2 treatment combined with magnetic fluid hyperthermia on Lewis lung cancer-bearing mice

    PubMed Central

    HU, RUNLEI; MA, SHENGLIN; KE, XIANFU; JIANG, HONG; WEI, DONGSHAN; WANG, WEI

    2016-01-01

    The present study aimed to investigate the therapeutic effect of interleukin-2 (IL-2) treatment combined with magnetic fluid hyperthermia (MFH) on Lewis lung cancer-bearing mice. Magnetic fluids were prepared in vitro and directly injected into the tumors in the mice, which were subjected to an alternating magnetic field. The temperature in the tumor reached 43C and was maintained by controlling the strength of magnetic field for 30 min. Twenty-four hours later, IL-2 was injected directly into the tumors. Mice were divided into four groups: Group I (control), II (MFH), III (IL-2) and IV (IL-2+MFH). The tumor grew gradually in groups II and IV (both P<0.05) compared to the control group. Histological analysis showed that the tumor cells underwent apoptosis and necrosis. Immunohistochemistry results demonstrated that heat-shock protein 70 and cluster of differentiation (CD) 8-positive and CD4-positive T cells were strongly expressed following hypothermia. Therefore, the present study provided evidence that IL-2 treatment combined with MFH improves the therapeutic effect on lung cancer-bearing mice. PMID:26870335

  20. mRNA

    PubMed Central

    Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

    2013-01-01

    Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and “naked mRNA” for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters. PMID:23291946

  1. Lack of association between interleukin-2 (IL-2) gene rs2069762 polymorphism and cancer risk: a meta-analysis

    PubMed Central

    Wang, Yafeng; Shu, Yun; Jiang, Heping; Sun, Bin; Ma, Zhiqiang; Tang, Weifeng

    2015-01-01

    Interleukin-2 (IL-2) is an important member of the cytokines that play critical roles in carcinogenesis. Many studies have investigated the association between IL-2 rs2069762 polymorphism and cancer risk; however, the results remain controversial. The aim of this study is to assess the correlation between IL-2 rs2069762 polymorphism and cancer risk. All eligible case-control studies accorded with criteria published up to March 30, 2015 were identified by searching Embase and PubMed databases. Association between IL-2 rs2069762 polymorphism and cancer risk was assessed by crude odds ratios (ORs) with 95% confidence intervals (CIs), respectively. Ten case-control studies from nine publications with 3095 cases and 4480 controls were included. Overall, IL-2 rs2069762 polymorphism was not associated with cancer risk in five genetic models (G vs. T: OR = 1.07, 95% CI = 0.95-1.21, P = 0.278; GG vs. TT: OR = 1.16, 95% CI = 0.86-1.57, P = 0.317; GG + TG vs. TT: OR = 1.09, 95% CI = 0.93-1.28, P = 0.273; GG vs. TT + TG: OR = 1.11, 95% CI = 0.85-1.44, P = 0.451; TG vs. TT: OR = 1.08, 95% CI = 0.92-1.28, P = 0.339, respectively). Similar results were also obtained after stratified by ethnicity and cancer type. This meta-analysis indicates that IL-2 rs2069762 T>G polymorphism is not associated with cancer risk. And the same conclusion is drawn after stratified by cancer type and ethnicity. PMID:26550166

  2. Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways.

    PubMed

    Doucette, Carolyn D; Greenshields, Anna L; Liwski, Robert S; Hoskin, David W

    2015-04-01

    Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease. PMID:25655587

  3. Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo.

    PubMed Central

    Spiegelberg, H L; Falkoff, R J; O'Connor, R D; Beck, L

    1991-01-01

    The effect of interleukin-2 (IL-2) on IL-4-induced IgE and IgG4 secretion by B cells in peripheral blood mononuclear cell (PBMC) preparations from non-atopic healthy humans and atopic dermatitis patients was investigated. PBMC were cultured at an optimal concentration of recombinant IL-4 with or without addition of IL-2 for 10 days. Native and recombinant IL-2 inhibited the IL-4-induced IgE and IgG4 secretion in a dose-dependent manner by cells from both normal and atopic donors. Rabbit antibodies to IL-2 or to the monoclonal anti-IL-2 receptor antibody anti-TAC reversed the IL-2 effect. Culturing cells with IL-4 and IL-2 for 1 or 2 days only slightly suppressed the IgE and IgG4 secretion whereas addition of IL-2 to IL-4 containing cultures on day 4 or 5 inhibited the IgE and IgG4 secretion more effectively. This is in contrast to interferon-gamma (IFN-gamma) which inhibited the IL-4 induced IgE and IgG4 secretion when added for the first 24 or 48 h but had no effect when added on days 4 or 5. The data demonstrate that both IL-2 and IFN-gamma act as antagonists in the IL-4-induced IgE and IgG4 secretion by human B cells; while IL-2 appears to inhibit relatively late in culture, IFN-gamma has an early inhibitory effect, suggesting that the two lymphokines inhibit the IL-4 effect by different mechanisms. PMID:1904325

  4. Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients.

    PubMed

    Rosenzwajg, Michelle; Churlaud, Guillaume; Mallone, Roberto; Six, Adrien; Dérian, Nicolas; Chaara, Wahiba; Lorenzon, Roberta; Long, S Alice; Buckner, Jane H; Afonso, Georgia; Pham, Hang-Phuong; Hartemann, Agnès; Yu, Aixin; Pugliese, Alberto; Malek, Thomas R; Klatzmann, David

    2015-04-01

    Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases. PMID:25634360

  5. Secretion of a unique peptide from interleukin-2-stimulated natural killer cells that induces endomitosis in immature human megakaryocytes.

    PubMed

    Angchaisuksiri, Pantep; Grigus, Shawn R; Carlson, Patricia L; Krystal, Geoffrey W; Dessypris, Emmanuel N

    2002-01-01

    When interleukin-2 (IL-2) was added to immature, low-ploidy (greater than 80% 2N+4N) megakaryocytes generated in IL-3 and stem cell factor (SCF)-containing liquid cultures of blood mononuclear cells highly enriched in hematopoietic progenitors, a 2- to 6-fold increase in the absolute number of polyploid (more than 8N) megakaryocytes was noted. This effect was found to be indirect and was mediated through natural killer (NK) cells that constitute the major lymphoid cell contaminating day 6 megakaryocyte cell populations. IL-2 had no effect on megakaryocytes generated from CD34(+) cells stimulated with IL-3 and SCF. However, medium conditioned by IL-2-stimulated, but not resting, NK cells (NKCM) contained a trypsin-sensitive factor capable of increasing 2- to 5-fold the number of polyploid megakaryocytes generated in vitro from IL-3 and SCF-stimulated CD34(+) cells. The activity in NKCM was dose dependent and could not be neutralized by an excess of antibodies to IL-6, IL-11, leukemia inhibitory factor (LIF), gp130, stromal cell derived factor-1a (SDF-1a), and thrombopoietin (TPO). Addition of IL-11, but not TPO, to NKCM-containing cultures resulted in further augmentation of polyploidy, with the generation of 50% to 70% polyploid megakaryocytes with a modal ploidy of 16N. This factor is distinct from TPO because it induces endomitosis in IL-3-generated megakaryocytes in vitro, whereas TPO does not, and its activity on megakaryocyte ploidy is not altered by optimal concentrations of TPO. In addition, no message for TPO is detectable in IL-2-stimulated NK cells by reverse transcription-polymerase chain reaction. These findings indicate that IL-2-stimulated NK cells produce a novel peptide, distinct from TPO, IL-6, IL-11, LIF, other gp130-associated interleukins, and SDF1a, that can induce in vitro endomitosis in immature human megakaryocytes in the presence of IL-3 and SCF. PMID:11756162

  6. Inhibition of interleukin-2-induced T-cell proliferation by sera from patients with the acquired immune deficiency syndrome.

    PubMed

    Donnelly, R P; Tsang, K Y; Galbraith, G M; Wallace, J I

    1986-01-01

    Sera from 22 patients with either lymphadenopathy syndrome (LAS), acquired immune deficiency syndrome (AIDS)-related complex (ARC), or acquired immune deficiency syndrome were examined for their effect on the interleukin-2 (IL-2)-induced proliferative response of an IL-2-dependent cytotoxic T-cell line, CTL-20. All of the patient sera included in this study were positive for the presence of antibodies against human T-cell lymphotropic virus type III (HTLV-III) as determined by an HTLV-III-specific enzyme-linked immunosorbent assay (ELISA). Eighteen of the 22 patient sera examined (81.8%) exhibited at least a modest suppressive effect on the proliferative response of CTL-20 cells. The inhibitory effect was dose-dependent and varied in intensity for each individual serum. In many cases, the magnitude of suppression was absolute in that it totally abrogated IL-2-induced DNA synthesis. Normal human serum (NHS) exerted no suppressive influence on the IL-2-induced proliferative response of identical control cultures. This same panel of 22 patient sera exhibited no significant inhibitory effects on the levels of protein synthesis in cultures of a non-IL-2-dependent human T-cell line, CCRF-HSB-2, indicating that the suppressive effect was not mediated by nonspecific serum cytotoxicity. The inhibitory effect of patient sera in the IL-2-dependent target cell assay correlated with the ability of these same sera to suppress the mitogen-induced proliferative response of normal human peripheral blood lymphocytes (PBL). These observations are particularly striking in view of the recognized defects of IL-2-dependent effector T-cell functions in AIDS. PMID:3007564

  7. Human and Mouse CD8+CD25+FOXP3+ Regulatory T Cells at Steady State and during Interleukin-2 Therapy

    PubMed Central

    Churlaud, Guillaume; Pitoiset, Fabien; Jebbawi, Fadi; Lorenzon, Roberta; Bellier, Bertrand; Rosenzwajg, Michelle; Klatzmann, David

    2015-01-01

    In addition to CD4+ regulatory T cells (Tregs), CD8+ suppressor T cells are emerging as an important subset of regulatory T cells. Diverse populations of CD8+ T cells with suppressive activities have been described. Among them, a small population of CD8+CD25+FOXP3+ T cells is found both in mice and humans. In contrast to thymic-derived CD4+CD25+FOXP3+ Tregs, their origin and their role in the pathophysiology of autoimmune diseases (AIDs) are less understood. We report here the number, phenotype, and function of CD8+ Tregs cells in mice and humans, at the steady state and in response to low-dose interleukin-2 (IL-2). CD8+ Tregs represent approximately 0.4 and 0.1% of peripheral blood T cells in healthy humans and mice, respectively. In mice, their frequencies are quite similar in lymph nodes (LNs) and the spleen, but two to threefold higher in Peyer patches and mesenteric LNs. CD8+ Tregs express low levels of CD127. CD8+ Tregs express more activation or proliferation markers such as CTLA-4, ICOS, and Ki-67 than other CD8+ T cells. In vitro, they suppress effector T cell proliferation as well as or even better than CD4+ Tregs. Owing to constitutive expression of CD25, CD8+ Tregs are 20- to 40-fold more sensitive to in vitro IL-2 stimulation than CD8+ effector T cells, but 24 times less than CD4+ Tregs. Nevertheless, low-dose IL-2 dramatically expands and activates CD8+ Tregs even more than CD4+ Tregs, in mice and humans. Further studies are warranted to fully appreciate the clinical relevance of CD8+ Tregs in AIDs and the efficacy of IL-2 treatment. PMID:25926835

  8. Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

    1996-04-01

    Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the C? 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

  9. Serum level of soluble interleukin-2 receptor correlates with CD25 expression in patients with T lymphoblastic lymphoma.

    PubMed

    Toji, Tomohiro; Takata, Katsuyoshi; Sato, Yasuharu; Miyata-Takata, Tomoko; Hayashi, Eiko; Habara, Toshiyuki; Maeda, Yoshinobu; Tanimoto, Mitsune; Yoshino, Tadashi

    2015-08-01

    Acute lymphoblastic leukaemia/lymphoma (ALL/LBL) is an aggressive form of non-Hodgkin's lymphoma (NHL) affecting B-cells or T-cells, respectively. The serum level of soluble interleukin-2 receptor (sIL-2R) is known to reflect the immune activity and tumour volume in aggressive NHL; however, the release of sIL-2R in LBL has not been extensively studied. Further, the relationship between sIL-2R release and the expression level of IL-2R ? subunit (CD25) remains unknown. In the present study, we examined the serum level of sIL-2R in 23 patients with T lymphoblactic lymphoma (T-LBL) and compared these with the levels in 20 patient with T acute lymphoblastic leukaemia (T-ALL), 40 patients with diffuse large B-cell lymphoma (DLBCL) and 40 patients with peripheral T-cell lymphoma (PTCL), not otherwise specified. The release of sIL-2R into the serum in patients with T-LBL was significantly lower than that for T-ALL, DLBCL and PTCL (p<0.001). Immunohistochemistry revealed that CD25 expression was correlated with the serum level of sIL-2R in T-LBL (p=0.0069), whereas no correlation was found to exist between serum sIL-2R levels and CD25 expression in patients with DLBCL (p=0.348) and PTCL (p=0.266). Furthermore, double immunohistochemical analysis revealed that CD25-positive cells were also found to be Foxp3-positive non-neoplastic T-cells. In conclusion, CD25-positive non-neoplastic T-cells in T-LBL are presumed to be the primary source of sIL-2R, and the low number of cells present results in a lower level of sIL-2R released into the serum compared with the other aggressive and highly aggressive lymphomas. PMID:25935549

  10. Enhancement of antibody-dependent cellular cytotoxicity of neonatal cells by interleukin-2 (IL-2) and IL-12.

    PubMed

    Nguyen, Q H; Roberts, R L; Ank, B J; Lin, S J; Lau, C K; Stiehm, E R

    1998-01-01

    Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture. PMID:9455889

  11. Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

    PubMed Central

    Nagaraj, Srinivas; Ziske, Carsten; Schmidt-Wolf, Ingo GH

    2004-01-01

    Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.61079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line. PMID:15329148

  12. Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors.

    PubMed Central

    Baxevanis, C. N.; Dedoussis, G. V.; Gritzapis, A. D.; Stathopoulos, G. P.; Papamichail, M.

    1994-01-01

    Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions. PMID:7917907

  13. Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours.

    PubMed

    Garca-Vzquez, M D; Boyano, M D; Caavate, M L; Gardeazabal, J; de Galdeano, A G; Lpez-Michelena, T; Ratn, J A; Izu, R; Daz-Ramn, J L; Daz-Prez, J L

    2000-12-01

    Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms. PMID:11125310

  14. Interleukin-2 from Adaptive T Cells Enhances Natural Killer Cell Activity against Human Cytomegalovirus-Infected Macrophages

    PubMed Central

    Wu, Zeguang; Frascaroli, Giada; Bayer, Carina; Schmal, Tatjana

    2015-01-01

    ABSTRACT Control of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV-seropositive donors showed an enhanced activity toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4+ T cells when reexposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV-infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMVnamely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunitybut also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness. IMPORTANCE Human cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host?s immunity becomes suppressed. Both innate immunity and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus-specific T cells are the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4+ T cells after reexposure to HCMV enhances the activity of NK cells in response to HCMV-infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act against HCMV infection. PMID:25855747

  15. Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1.

    PubMed Central

    Hutchings, D L; Campos, M; Qualtiere, L; Babiuk, L A

    1990-01-01

    The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia. PMID:2166810

  16. Lectin interactions with the Jurkat leukemic T-cell line: quantitative binding studies and interleukin-2 production

    SciTech Connect

    Dupuis, G.; Bastin, B.

    1988-03-01

    Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity (Kass (apparent) approximately equal to 9 x 10(9) M-1), followed by pea lectin and WGA (Kass (apparent) approximately equal to 3 x 10(9) M-1). The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 (high-affinity sites) and (apparent) K2 = 2.6 x 10(9) M-1 (low-affinity sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml).

  17. Low-dose inhalation of interleukin-2 bio-chemotherapy for the treatment of pulmonary metastases in melanoma patients

    PubMed Central

    Posch, C; Weihsengruber, F; Bartsch, K; Feichtenschlager, V; Sanlorenzo, M; Vujic, I; Monshi, B; Ortiz-Urda, S; Rappersberger, K

    2014-01-01

    Background: Interleukin-2 (IL-2) treatment for patients with metastatic melanoma has shown remarkable durable responses. Systemic administration of IL-2 may cause severe side effects, whereas local administration is considered to be a safe alternative. The lungs are common sites of metastases in melanoma patients causing considerable respiratory problems. We sought to evaluate the potential antitumoral effect of a low-dose inhalative IL-2 (lh-IL-2) regimen for patients with melanoma lung metastases. In addition, we explored the prophylactic potential of Ih-IL-2 after surgical removal of lung metastases in a study carried out in an outpatient setting. Methods: Twenty patients with American Joint Committee on Cancer stage-IV (M1b and M1c) melanoma were enrolled in this study and treated with 3 3 million IU inhalative IL-2 q.d. together with monthly dacarbazine bolus injections. Five patients received lh-IL-2 after surgical resection of lung metastases to prevent recurrence of the disease (prophylaxis group, N=5). All other patients were enrolled in the treatment group (N=15). Clinical evaluations were carried out monthly and radiological follow-up was performed every third month. Results: Nine patients in the treatment group had a clinical benefit with partial regression (27%) or stable disease (33%). Four patients had progression of lung metastases (26.7%) and two patients were not evaluable (13.3%). In the prophylaxis group, none of the patients developed new lung metastases during lh-IL-2 therapy. The median follow-up period was 7.8 months in the treatment group and 25.7 months in the prophylaxis group. In the majority of patients, treatment was well tolerated. Conclusions: Low-dose IL-2 inhalation might offer an effective and safe treatment option for lung metastases in melanoma patients. In addition, lh-IL-2 may have a prophylactic potential to prevent recurrence in the lungs after pulmonary melanoma metastasectomy. Administration can easily be performed in an outpatient setting, thus offering an attractive treatment option. PMID:24518593

  18. Low CD3+CD28-induced interleukin-2 production correlates with decreased reactive oxygen intermediate formation in neonatal T cells.

    PubMed

    Kilpinen, S; Hurme, M

    1998-06-01

    The capacity of neonatal T cells to secrete interleukin-2 (IL-2) has been reported to be variable. We analysed IL-2 production in purified neonatal and adult T cells using polyclonal activator phorbol ester + calcium ionophore (PDBu + iono) or receptor-mediated anti-CD3/anti-CD3+ anti-CD28 stimulation. PDBu + iono induced equally high IL-2 levels in both groups and, when stimulated with plate-bound anti-CD3 monoclonal antibody (mAb), the IL-2 secretion by neonatal cells was undetectable and adult cells produced low amounts of IL-2 (mean 331 +/- 86 pg/ml). The addition of anti-CD28 mAb to anti-CD3-stimulated cells markedly increased IL-2 production in both cell types, but levels of IL-2 in neonatal T cells remained clearly lower than those of adult T cells (respective mean values: 385 +/- 109 pg/ml and 4494 +/- 1199 pg/ml). As NF-kappa B is a critical transcription factor in the control of IL-2 expression, we next analysed its nuclear translocation in neonatal and adult T cells using the electrophoretic mobility shift assay and, because induction of reactive oxygen intermediates (ROI) is required for the activation of NF-kappa B, we also analysed levels of intracellular ROI in these cells using the ROI-reactive fluorochrome DCFH-DA and flow cytometry. In neonatal T cells NF-kappa B activation and ROI formation after anti-CD3 stimulation were low compared with adult T cells and, although addition of anti-CD28 mAb increased induction of NF-kappa B and ROI formation, levels similar to those of adults were not achieved. After PDBu + iono stimulation, the cells showed similar ROI formation and IL-2 secretion. Our results suggest that reduced IL-2 production by neonatal T cells is specific for anti-CD3 and anti-CD3+ anti-CD28-mediated stimulation and that these activators cannot effectively activate the ROI-NF-kappa B signalling pathway in neonatal T cells. PMID:9741337

  19. Annual Hospital Volume of High Dose Interleukin-2 and Inpatient Mortality in Melanoma and Renal Cell Carcinoma Patients

    PubMed Central

    Mehta, Kathan; Appleman, Leonard; Wang, Hong; Tarhini, Ahmad A.; Parikh, Rahul A.

    2016-01-01

    Background Immunotherapy using high dose interleukin-2 (HD IL2) in patients with renal cell carcinoma (RCC) and melanoma is associated with severe toxicities. The association between annual hospital volume of HD IL2 and inpatient mortality is not well studied. In this study we aim to quantify the impact of annual hospital volume of HD IL2 on inpatient mortality using National Inpatient Sample (NIS) data. Methods We did a cross-sectional study using NIS, one of the largest inpatient datasets in United States, from 2003 to 2011. Patients with melanoma and RCC receiving HD IL2 were identified by ICD9 procedure code 00.15. The primary outcome was inpatient mortality. Using Joinpoint regression, which detects change in trend of inpatient mortality with change in annual volume, the hospitals were classified in three volume categories (low: 1–40, medium: 41–120, high: >120). Multivariate logistic regression was used to identify predictors of inpatient mortality controlling for confounders. Results From 2003 to 2011, 29,532 patients with RCC or melanoma who received HD IL2 were identified, and 124 died during the hospitalization (0.4%). The hospitals with low, medium and high annual volume had significant difference in inpatient mortality (0.83%, 0.29% and 0.13% respectively, p = 0.0003). On multivariate analysis, low volume hospitals were associated with significantly higher odds of inpatient mortality (OR 6.1, 95% CI 1.6–23.2, p = 0.003) as compared to high volume hospitals. Additionally, the hospitals with annual volume of 1–20 had even higher rates (1.31% vs. 0.13%, p<0.0001) and multivariate odds (OR 8.9, 95% CI 2.4–33.2, p = 0.0006) of inpatient mortality as compared to high volume hospitals. Conclusions Lower annual hospital volume of HD IL2 is associated with worse outcomes. Annual hospital volume of 1–40 and 1–20 treatments per year is associated with 6 and 9 times higher odds of inpatient mortality respectively as compared to high volume hospitals. Our findings provide preliminary evidence for a volume-outcome relationship for RCC and melanoma patients undergoing HD IL2 treatment. They support future volume-outcome analyses in relation to other anti-cancer therapies that require special training and expertise. PMID:26799322

  20. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    SciTech Connect

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. )

    1990-08-15

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

  1. Subcutaneous administration of interleukin-2 triggers Fcgamma receptor I expression on human peripheral blood neutrophils in solid and hematologic malignancies.

    PubMed

    Sconocchia, G; Cococcetta, N Y; Campagnano, L; Amadori, S; Iorio, B; Boffo, V; Ferdinandi, V; Del Principe, I; Adorno, D; Casciani, C U

    2001-01-01

    Freshly isolated human polymorphonuclear cells (PMNCs) constitutively express Fcgamma receptor (Fc-gammaR) II and FcgammaRIII on the cell surface but not FcgammaRI. Cytokines such as interferon-gamma (IFNgamma), granulocyte-macrophage colony-stimulating factor (CSF), and granulocyte-CSF trigger FcgammaRI expression on (PMNCs). Because PMNCs express interleukin (IL)-2 receptor, we investigated whether IL-2 can induce FcgammaRI expression on PMNCs isolated from IL-2-treated metastatic renal cell carcinoma (MRCC) and low-grade non-Hodgkin lymphoma (LGNHL) patients. Pretherapy flow cytometry analysis of Fcgamma receptors on PMNCs did not show FcgammaRI expression. Interestingly, 3 days after therapy, PMNCs displayed a detectable amount of FcgammaRI on the cell surface. Kinetic studies on the in vivo effects of IL-2 on MRCC patients showed that FcgammaRI was transiently expressed, starting within 3-6 days of therapy, remaining expressed for 10-15 days, and rapidly declining, whereas such expression remained stable for months in LGNHL patients. In contrast, Fc-gammaRII was not affected. In addition, FcgammaRI+ PMNCs coated in vitro with a bispecific antibody Fab anti-FcgammaRI x anti-HER-2/neu formed intercellular conjugates with a human HER-2/neu-transfected 3T3 cell line (HER-2/neu-3T3). Interleukin-2 treatment increased the number of FcgammaRIII low eosinophils, leading to a change in FcgammaRIII distribution among granulocyte cell subsets. In vitro IL-2 treatment of purified PMNCs failed to generate Fc-gammaRI expression, suggesting that IL-2 indirectly causes FcgammaRI expression. During the IL-2 administration, we did not observe significant changes in IFNgamma serum level. In conclusion, our observation may be used to potentiate the antitumor effects of IL-2 in novel immunotherapy regimens, perhaps by redirecting FcgammaRI+ PMNCs against cancer cells by heteroconjugate antibodies and monitoring the biologic activity of subcutaneous IL-2 in cancer patients. PMID:11565839

  2. In vivo administration of interferon alpha and interleukin 2 induces proliferation of lymphoid cells in the organs of mice

    SciTech Connect

    Puri, R.K.; Travis, W.D.; Rosenberg, S.A. )

    1990-09-01

    We have previously shown that interleukin 2 (IL-2) synergizes with interferon alpha (IFN-alpha) in mediating the regression of established pulmonary and hepatic metastases and the reduction of intradermal tumor in various murine tumor models. To understand the mechanism of synergy, we have examined lymphoid cell proliferation in various organs of mice in response to IL-2 and IFN-alpha administration. We have utilized a technique for labeling newly synthesized DNA in vivo with 5-(125I)iodo-2'-deoxyuridine to examine proliferation of endogenous cells in response to IL-2 and IL-2 plus IFN-alpha. A proliferation index was calculated by dividing cpm in the tissues treated with cytokines by cpm obtained in corresponding tissues of control mice. After 4 days of IL-2 administration, a significant uptake of 5-(125I)iodo-2'-deoxyuridine was observed in the lungs, liver, kidneys, and spleen (proliferation index of 13, 10.3, 3.6, and 3.2, respectively). IFN-alpha alone mediated very little incorporation of radiolabel but when administered in combination with IL-2 a reduction of IL-2-induced proliferation was seen on day 4. For example 19,272 +/- 4,556 cpm (mean +/- SE) were obtained in the liver of IL-2-treated mice, compared to 8,103 +/- 2,111 cpm in livers of IL-2 plus IFN-alpha-treated mice (P less than 0.05). Similar inhibition of IL-2-induced proliferation was observed in the lungs, kidneys, and spleen. In contrast, on days 7 or 8, higher uptake of radiolabel was obtained in IFN-alpha plus IL-2-treated lungs, liver, and kidneys, compared to organs of mice treated with IL-2 alone or IFN-alpha alone. A proliferation index of 30.5, 9.8, and 10 was obtained in the lungs, liver, and kidneys of IL-2- plus IFN-alpha-treated animals, compared to 9.6, 3.6, and 5.5 in the corresponding organs of IL-2-treated mice.

  3. The relationship between thyroid function, serum monokine induced by interferon gamma and soluble interleukin-2 receptor in thyroid autoimmune diseases

    PubMed Central

    Jiskra, J; Antoov, M; Lmanov, Z; Teli?ka, Z; Lacinov, Z

    2009-01-01

    Interactions between cytokines play an important role in the development of thyroid autoimmunity. Using enzyme-linked immunosorbent assay we investigated serum concentrations of soluble interleukin-2 receptor (sIL-2R), interferon-gamma, tumour necrosis factor (TNF)-?, interleukin (IL)-10, CD30, monokine induced by interferon-gamma (MIG), cytotoxic T lymphocyte antigen-4 and markers of apoptosis decoy receptor 3 and Bcl-2 in 28 patients with hyperthyroid Graves' disease (GD), 24 patients with untreated Hashimoto's thyroiditis (HT) and 15 healthy controls. TNF-?, IL-10 and sIL-2R were higher in GD compared with HT and controls (TNF-?: 879 in GD versus 254 pg/ml in HT, P = 001; IL-10: 1000 versus 310 versus 310 pg/ml, P1 < 0001, P2 = 0005; sIL-2R: 126 versus 064 versus 046 ng/ml, P < 0001). MIG and CD30 were higher in HT compared with controls (64922 26255 versus 31295 14335 pg/ml, P = 0037, 657 235 versus 303 104 U/ml, P = 0036 respectively). In GD sIL-2R decreased when the euthyroid state was achieved (131 064 versus 0260 011, n = 12, P < 0001). sIL-2R correlated positively with free thyroxine (FT4) (R = 0521, P = 0000) and negatively with thyroid stimulating hormone (TSH) (R = ?0472, P = 000132). MIG correlated negatively with FT4 (R = ?0573, P = 000234) and positively with TSH (R = 0462, P = 00179). The results suggest that serum concentrations of sIL-2R and MIG are related to thyroid function rather than to activation of autoimmunity. PMID:19250272

  4. Adoptive immunotherapy of human pancreatic cancer with lymphokine-activated killer cells and interleukin-2 in a nude mouse model

    SciTech Connect

    Marincola, F.M.; Da Pozzo, L.F.; Drucker, B.J.; Holder, W.D. Jr. )

    1990-11-01

    A pancreatic cancer cell line was grown in orthotopic and heterotopic positions in young Swiss/NIH nude mice, which were tested with adoptive immunotherapy. Mice were injected with 1 x 10(7) human cancer cells in the subcutaneous tissue and duodenal lobe of the pancreas. The mice were randomly divided into four groups: group IA (LAK + IL-2) (N = 25) received 2 X 10(7) human lymphokine-activated killer (LAK) cells from normal donors by tail vein injection followed by 10,000 units of human recombinant interleukin-2 (IL-2) given intraperitoneally every 12 hours for 28 days; group IB (IL-2) (N = 27) was given the same dose of IL-2 alone; group IC (RPMI-1640) (N = 18) received a placebo consisting of 1 ml of RPMI-1640 intraperitoneally every 12 hours; and group ID (LAK) (N = 14) received 2 X 10(7) LAK cells but no IL-2. Toxicity was significantly higher in group IB, with a mortality rate of 45.5% (10/22 animals) versus a 0% mortality (0/25) in group IA. None of the group IA or IB animals died of pancreatic cancer during the experiment. The animals that did not receive IL-2 died before 28 days in 14.2% of group IC and in 16.7% of group ID. The area under the growth curve of subcutaneous tumors during the course of treatment and the pancreatic tumor weight at the end of treatment were compared in each group. Subcutaneous tumors had a reduced rate of growth in group IA animals compared to all the other treatments. Pancreatic tumor growth was slowed in group IA. The animals treated with IL-2 alone (group IB) showed some slowing of tumor growth that was intermediate between group IA, group IC, and group ID. A similar experiment was done with irradiated (375 rad) mice. Nine nude mice with tumors were treated with LAK + IL-2 (group IIA), eight received IL-2 alone (group IIB), and seven received placebo (group IIC).

  5. Immunotherapy (recombinant interleukin 2), hormone therapy (medroxyprogesterone acetate) and antioxidant agents as combined maintenance treatment of responders to previous chemotherapy.

    PubMed

    Mantovani, G; Maccio, A; Madeddu, C; Massa, E; Mudu, M C; Mulas, C; Gramignano, G; Massidda, S; Murgia, V; Lusso, M R; Mura, L

    2001-02-01

    An open, non-randomized phase II study was carried out including all patients treated with whatever chemotherapy or combined modality regimen for whatever cancer who were in clinical objective response or stable disease (SD) for more than three months, to receive maintenance treatment with recombinant interleukin-2 (rIL-2) plus medroxyprogesterone acetate (MPA) plus antioxidant agents alpha-lipoic acid (ALA) and N-acetyl cysteine (NAC). The main study endpoints were clinical outcome and toxicity. The secondary endpoints were effects of treatment on cancer-related anorexia/cachexia syndrome (CACS) symptoms, on serum levels of proinflammatory cytokines, IL-2, C-reactive protein (CRP) and leptin as well as the evaluation of quality of life (QL). rIL-2 was administered at a dose of 1.8 MIU subcutaneously three times/week on alternate days for the first two weeks of every month and MPA was given orally at a dose of 500 mg once a day at alternate days without interruption. ALA 300 mg/day orally and NAC 1800 mg/day orally were also administered. The treatment was administered until progression of disease or appearance of toxicity. From July 1998 to May 2000, 16 patients were enrolled in the study (M/F ratio: 15/1; mean age: 62 years, range 45-71). The median duration of maintenance treatment was 10 months (range 5-22). The response to maintenance treatment at September 2000 was: CR (persistent throughout the treatment) 4 patients (25%); SD 1 patient (6.2%); PD 11 patients (68.8%). The median duration of response was 9.8 months (range: 5-22+). The median follow-up duration was 19 months (range: 8-102). The median OS was not reached. The median PFS was 14 months (range 1-29). The 1-year survival rate was 25%. At September 2000, 9 patients are still surviving. No grade 3/4 toxicity was observed. One Grade 2 skin toxicity was observed and Grade 1: 2 fever, 2 thrombocytopenia, 1 neutropenia and 1 skin were observed. The ECOG PS did worsen significantly, the body weight and BMI increased significantly after treatment, whereas the appetite did not change significantly. The QL evaluation showed a significant amelioration of cognitive functions and a borderline significant amelioration of emotional functions after treatment, whereas a borderline worsening of dyspnea was observed. The absolute lymphocyte count increased significantly after the maintenance treatment, as well as the serum IL-2, TNFalpha decreased at borderline statistical significance; the serum levels of leptin did not change significantly. The evaluation of patient subgroups showed that responders/survivors had a pattern superimposable to that of whole patient population, the patients who rapidly progressed and died exhibited no significant changes of these parameters during treatment. The results of the present study suggest that the host immune response, evaluated by several parameters, after IL-2 administration, (e.g. lymphocytosis), are worth further study as potential markers for the major end points of cancer treatment, i.e. OS and QL, in an adequate number of patients. PMID:11172608

  6. Treatment of advanced renal cell cancer with sequential intravenous recombinant interleukin-2 and subcutaneous alpha-interferon.

    PubMed

    Besana, C; Borri, A; Bucci, E; Citterio, G; Di Lucca, G; Fortis, C; Matteucci, P; Tognella, S; Tresoldi, M; Baiocchi, C

    1994-01-01

    Starting from in vitro studies suggesting synergistic antitumour activity against renal cell cancer (RCC) of recombinant interleukin-2 (rIL-2) and alpha-interferon (IFN), a phase II trial was initiated to test the clinical activity of this combination. The two cytokines were administered sequentially, with the aim of reducing the risk of additive toxicity and enhancing the immunological reaction against the tumour. The original treatment schedule consisted of rIL-2 18 x 10(6) U/m2/day by continuous intravenous infusion for 120 h days 1-5, and alpha-IFN 2b, at a flat dose of 9 x 10(6) U by subcutaneous or intramuscular injection thrice in a week, from day 8 to 28. Treatment was planned to be continued for six or more 28-day cycles, depending on clinical response. 12 patients were treated according to this schedule; as some cardiovascular toxicity was experienced in this set of patients, 11 further patients were treated with half-dose rIL-2 (i.e. 9 x 10(6) U/m2/day). 17 out of 23 enrolled patients completed at least one cycle of treatment and were evaluated for response. We observed six major responses [one complete response (CR) + five partial responses (PR)] for an objective response rate of 35% [95% confidence interval (CI) 17-59%]. 5 additional patients achieved stabilisation of disease; one of them reached CR after surgical extirpation of a lung mass. Sites of response included lung, nodes and bone. Duration of response is 12+ months for CR; 17, 16, 12+, 9 and 9 months for PRs. Median survival is 16 months. Response was not significantly different between full-dose and half-dose rIL-2. Considering stable disease (SD) as responses, there seemed to be a higher chance of response for patients with smaller tumour burden (P = 0.032). The toxicity of rIL-2 treatment, mainly cardiovascular, was substantial; 9 patients experienced severe cardiotoxicity, consisting of major arrhythmias, myocardial ischaemia, reduction of ejection fraction measured with heart radionuclide scan, and were excluded from continuing treatment. Other rIL-2-related toxicities forcing exclusion from the study were severe thrombocytopenia (1 case), and generalised exfoliative dermatitis requiring steroids (1 case). Otherwise, treatment was well tolerated; rIL-2-related toxicities promptly recovered after rIL-2 discontinuation in the majority of cases, and no treatment-related deaths were reported. The half-dose rIL-2 regimen was significantly less toxic in terms of hypotension (P = 0.014), fever (P = 0.014), oliguria (P = 0.042), serum creatinine elevation (P = 0.009) and prothrombin time elongation (P = 0.038).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7999416

  7. An Anti-Interleukin-2 Receptor Drug Attenuates T- Helper 1 Lymphocytes-Mediated Inflammation in an Acute Model of Endotoxin-Induced Uveitis

    PubMed Central

    Navea, Amparo; Almansa, Inmaculada; Muriach, Mara; Bosch-Morell, Francisco

    2014-01-01

    The aim of the present study was to evaluate the anti-inflammatory efficacy of Daclizumab, an anti-interleukin-2 receptor drug, in an experimental uveitis model upon a subcutaneous injection of lipopolysaccharide into Lewis rats, a valuable model for ocular acute inflammatory processes. The integrity of the blood-aqueous barrier was assessed 24 h after endotoxin-induced uveitis by evaluating two parameters: cell count and protein concentration in aqueous humors. The histopathology of all the ocular structures (cornea, lens, sclera, choroid, retina, uvea, and anterior and posterior chambers) was also considered. Enzyme-linked immunosorbent assays of the aqueous humor samples were performed to quantify the levels of the different chemokine and cytokine proteins. Similarly, a biochemical analysis of oxidative stress-related markers was also assessed. The inflammation observed in the anterior chamber of the eyes when Daclizumab was administered with endotoxin was largely prevented since the aqueous humor protein concentration substantially lowered concomitantly with a significant reduction in the uveal and vitreous histopathological grading. Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon-?, also significantly reduced with related anti-oxidant systems recovery. Daclizumab treatment in endotoxin-induced uveitis reduced Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon gamma, by about 6070% and presented a preventive role in endotoxin-induced oxidative stress. This antioxidant protective effect of Daclizumab may be related to several of the observed Daclizumab effects in our study, including IL-6 cytokine regulatory properties and a substantial concomitant drop in INF?. Concurrently, Daclizumab treatment triggered a significant reduction in both the uveal histopathological grading and protein concentration in aqueous humors, but not in cellular infiltration. PMID:24595020

  8. Induction of myasthenia gravis, myositis, and insulin-dependent diabetes mellitus by high-dose interleukin-2 in a patient with renal cell cancer.

    PubMed

    Fraenkel, Paula G; Rutkove, Seward B; Matheson, Jean K; Fowkes, Mary; Cannon, Marie E; Patti, Mary-Elizabeth; Atkins, Michael B; Gollob, Jared A

    2002-01-01

    Interleukin-2 is an effective agent against renal cell carcinoma and melanoma, but it has been associated with autoimmune sequelae such as hypothyroidism and vitiligo. A 64-year-old man with non-insulin-dependent diabetes and metastatic renal cell carcinoma developed insulin-dependent diabetes after his first cycle of therapy with high-dose (HD) interleukin-2. After additional therapy with interleukin-2, the patient developed generalized myasthenia gravis (MG) and polymyositis, both of which responded to treatment with corticosteroids and plasmapheresis. To investigate the role of IL-2 in the development of these autoimmune complications, autoantibody titers were assayed from serum obtained before and after IL-2 treatment and after treatment with corticosteroids plus plasmapheresis. Before IL-2 treatment, the patient had antibodies directed against insulin, islet cell antigens, and striated muscle. Acetylcholine receptor antibody levels were normal before starting IL-2. After treatment with IL-2, the patient developed acetylcholine receptor binding antibodies and exhibited an increase in the striated muscle antibody titer from 1:40 to 1:160. Recovery from the MG and polymyositis was associated with substantial decreases in the acetylcholine receptor and striated muscle antibody titers. These findings suggest that HD IL-2 accelerated the progression of latent autoimmune diabetes and myositis in this patient whose tolerance to islet cell antigens and striated muscle had already been broken and precipitated a break in tolerance to the acetylcholine receptor resulting in the development of MG. This case demonstrates the importance of prompt recognition of IL-2-induced MG and shows how this complication can be successfully managed with aggressive therapy. PMID:12142560

  9. Effect in vitro of a bacterial extract (OM-89) on interleukin 1 and interleukin 2 production by peripheral blood mononuclear cells from healthy subjects and rheumatoid arthritis patients.

    PubMed

    Clot, J; Andary, M

    1990-01-01

    The effect of a lyophilized extract from Escherichia coli strains (OM-89) on interleukin 1 and interleukin 2 production was studied by using peripheral blood mononuclear cells (PBMC) from healthy volunteers and from patients suffering from rheumatoid arthritis (RA) since, in this autoimmune disease, an abnormal cytokine network has been already described. The secretion of interleukin 1 (IL-1) was investigated in supernatants of monocytes purified by adherence, and measured by the C3H/HeJ thymocyte co-mitogenic assay. OM-89 was able to induce the secretion of IL-1 by normal and RA monocytes to about half of the level reached when the same cells were stimulated by lipopolysaccharide. The production of interleukin 2 (IL-2) was investigated in supernatants of PBMC, stimulated or not by phytohaemagglutinin (PHA) and mixed or not with various concentrations of OM-89. The level of IL-2 in supernatants, as measured by the stimulation of the CTLL2 murine cell line, was lower in RA supernatants than in control ones. In the presence of PHA and OM-89, the IL-2 production was enhanced and normalized in supernatants from RA patients. Such data may help to explain the clinical improvement previously reported in RA patients orally treated with OM-89. PMID:2292468

  10. Release of IgD-binding factor by T cells under the influence of interleukin 2, interleukin 4, or cross-linked IgD.

    PubMed Central

    Amin, A R; Coico, R F; Finkelman, F; Siskind, G W; Thorbecke, G J

    1988-01-01

    Helper T cells with receptors specific for IgD have immunoaugmenting properties. We have now detected soluble IgD-binding factor in cell supernatants immobilized on nitrocellulose paper by their ability to bind 125I-labeled IgD. IgD-binding factor is released by normal splenic T cells stimulated with recombinant interleukin 2, recombinant interleukin 4, or crosslinked IgD in amounts paralleling the induction of IgD receptors on the cells. IgD receptors are constitutively produced by antigen-specific helper T-cell hybridomas 2H10 and A3.4C6. Incubation of these hybridoma cells with recombinant interleukin 2 increases release of IgD-binding factor while reducing expression of IgD receptors. Specificity of the binding factor for IgD is established by (i) competitive inhibition; (ii) the ability of the binding factor to bind radiolabeled IgD and not monoclonal IgE, IgG2a, or polyclonal IgG; and (iii) the removal of the binding factor on passage through an IgD-Sepharose column and recovery in a subsequent acid eluate. Images PMID:2973608

  11. Natural killer T cells constitutively expressing the interleukin-2 receptor ? chain early in life are primed to respond to lower antigenic stimulation.

    PubMed

    Ladd, Mihoko; Sharma, Ashish; Huang, Qing; Wang, Adele Y; Xu, Lixin; Genowati, Indira; Levings, Megan K; Lavoie, Pascal M

    2010-10-01

    Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interleukin-2 receptor ? chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25-expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krppel-like factor-2, a transcription factor associated with quiescent T cells, and require de novo T-cell receptor and CD28 co-stimulation to proliferate. In contrast to bona fide CD4/CD25-expressing regulatory T cells, neonatal iNKT cells do not suppress T-cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d-restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin-2 suggest that constitutive expression of CD25 'primes' neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25-expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT-cell repertoire early in life. PMID:20545784

  12. B7-H4 expression and its role in interleukin-2/interferon treatment of clear cell renal cell carcinoma

    PubMed Central

    XU, YIPENG; ZHU, SHAOXING; SONG, MEI; LIU, WEIHUI; LIU, CHENGYI; LI, YONGSHENG; WANG, MIN

    2014-01-01

    The immunological mechanism mediated by T cells is the main therapeutic target in the treatment of renal cell carcinoma (RCC) with interleukin (IL)-2 and interferon (IFN)-α. The aim of the present study was to evaluate the role of B7-H4 in the IL-2, IFN-α and IFN-γ treatment of clear cell RCC (ccRCC). A total of 154 paraffin-embedded ccRCC tissues were studied using immunohistochemistry, which subsequently indicated that positive B7-H4 expression is associated with adverse clinical features in ccRCC. The effects of IL-2, IFN-α and IFN-γ on B7-H4 expression in a ccRCC cell line were evaluated at the mRNA and protein levels. In addition, the effect of B7-H4 on the killing activity of T cells was detected. B7-H4 expression was identified to be upregulated by IL-2, IFN-α and IFN-γ, of which, IFN-γ was the most capable. Additionally, blocking of B7-H4/B7-H4 ligand interactions may rescue the killing activity of T cells. Altogether, the observations of the current study showed that the immune escape pathway induced by B7-H4 may be one of the most important reasons for the low efficacy of IL-2 and IFN-α and the inability to observe the efficacy of IFN-γ in mRCC. This indicates that B7-H4 may be used as a new molecular biology marker to select treatment options for patients with ccRCC. PMID:24765159

  13. Proliferative response of Tax1-transduced primary human T cells to anti-CD3 antibody stimulation by an interleukin-2-independent pathway.

    PubMed Central

    Akagi, T; Shimotohno, K

    1993-01-01

    The growth properties of human T-cell leukemia virus Tax1-transduced primary human T cells derived from peripheral blood lymphocytes were compared with those of the same subset of T cells transduced with a control vector. Tax1-transduced T cells exhibited slightly elevated responsiveness to externally added interleukin-2 (IL-2) and a markedly higher proliferative response to stimulation with anti-CD3 antibody. The proliferation after anti-CD3 antibody stimulation was mainly via an IL-2-independent pathway. Therefore, some other mechanism than the previously proposed IL-2 autocrine model seems to be involved in the process of deregulation of T-cell proliferation by Tax1. Moreover, Tax1-transduced T cells have continued to proliferate in medium containing IL-2 long after control T cells ceased to grow, and so they are considered to be immortalized. Images PMID:8437212

  14. Thymic regulatory T cell niche size is dictated by limiting interleukin 2 from antigen-bearing dendritic cells and feedback competition

    PubMed Central

    Weist, Brian M.; Kurd, Nadia; Boussier, Jeremy; Chan, Shiao Wei; Robey, Ellen A.

    2015-01-01

    Thymic regulatory T (Treg) cell production requires interleukin 2 (IL-2) and agonist TCR ligands, and is controlled by competition for a limited developmental niche, but the thymic sources of IL-2 and the factors that limit access to the niche are poorly understood. Here we show that IL-2 produced by antigen-bearing dendritic cells plays a key role in Treg cell development, and that existing Treg cells limit new Treg cell development by competing for IL-2. . Our data suggest that antigen-presenting cells that can provide both IL-2 and a TCR ligand comprise the thymic niche, and that competition by existing Treg cells for a limited supply of IL-2 provides negative feedback for new Treg cell production. PMID:25939026

  15. Partial agonist/antagonist mouse interleukin-2 proteins indicate that a third component of the receptor complex functions in signal transduction.

    PubMed Central

    Zurawski, S M; Imler, J L; Zurawski, G

    1990-01-01

    Some mouse interleukin-2 (mIL-2) proteins with substitutions at residue Gln141 are unable to trigger a maximal biological response. The Asp141 protein induces the lowest maximal response. The Asp141 protein can weakly antagonize the biological activity of mIL-2 and strongly antagonizes the biological activity of active mIL-2 mutant proteins that have defects in interactions with the high affinity receptor. Residue 141 mutant proteins bind with reduced affinity to T cells expressing the high affinity IL-2 receptor, yet bind normally to transfected fibroblasts expressing only the alpha and beta chains of the receptor. These results suggest that a third receptor component is important for both binding and signal transduction. PMID:2249656

  16. Discovery and structure-activity relationship of 3-aminopyrid-2-ones as potent and selective interleukin-2 inducible T-cell kinase (Itk) inhibitors.

    PubMed

    Charrier, Jean-Damien; Miller, Andrew; Kay, David P; Brenchley, Guy; Twin, Heather C; Collier, Philip N; Ramaya, Sharn; Keily, Shazia B; Durrant, Steven J; Knegtel, Ronald M A; Tanner, Adam J; Brown, Kieron; Curnock, Adam P; Jimenez, Juan-Miguel

    2011-04-14

    Interleukin-2 inducible T-cell kinase (Itk) plays a role in T-cell functions, and its inhibition potentially represents an attractive intervention point to treat autoimmune and allergic diseases. Herein we describe the discovery of a series of potent and selective novel inhibitors of Itk. These inhibitors were identified by structure-based design, starting from a fragment generated de novo, the 3-aminopyrid-2-one motif. Functionalization of the 3-amino group enabled rapid enhancement of the inhibitory activity against Itk, while introduction of a substituted heteroaromatic ring in position 5 of the pyridone fragment was key to achieving optimal selectivity over related kinases. A careful analysis of the hydration patterns in the kinase active site was necessary to fully explain the observed selectivity profile. The best molecule prepared in this optimization campaign, 7v, inhibits Itk with a K(i) of 7 nM and has a good selectivity profile across kinases. PMID:21391610

  17. Interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) cells and their precursors express the VGO1 antigen

    SciTech Connect

    Denegri, J.F.; Peterson, J.; Tilley, P. )

    1989-07-01

    Precursor and effector cells of recombinant interleukin-2 (r-IL-2)-induced lymphokine-activated killer (LAK) activity were investigated for their expression of VGO1. Peripheral blood lymphocytes (PBL) from normal donors were purified and separated in a FACS 420 into VGO1+- and VGO1- cell fractions before and after culture for 96 hr with 100 U/ml of r-IL-2. Their lytic activity against K 562 and Daudi cells was measured in a 51Cr release assay. The majority, if not all, of the LAK effector and precursor cells was VGO1+ lymphocytes. The expression of VGO1 by LAK precursor cells remained stable under the culture conditions used in our experiments. VGO1- lymphocytes cultured with r-IL-2 demonstrated neither LAK-induced activity nor expression of VGO1 antigen.

  18. Prolonged survival of a patient affected by pancreatic adenocarcinoma with massive lymphocyte and dendritic cell infiltration after interleukin-2 immunotherapy. Report of a case.

    PubMed

    Nobili, Cinzia; Degrate, Luca; Caprotti, Roberto; Franciosi, Claudio; Leone, Biagio Eugenio; Trezzi, Rosangela; Romano, Fabrizio; Uggeri, Fabio; Uggeri, Franco

    2008-01-01

    Several studies have shown that there is a paucity of immune cells within the stroma of pancreatic adenocarcinoma, a very aggressive cancer with a median survival of about 18 months. A 65-year-old man presented with jaundice. Abdominal ultrasound revealed intra- and extrahepatic bile duct dilatation and a 45-mm diameter hypoechoic solid mass within the pancreatic head; a computed tomography scan excluded vascular infiltration and metastatic lesions. The patient received immunotherapy consisting of 6,000,000 IU human recombinant interleukin-2 administered subcutaneously twice a day for 3 consecutive days. Thirty-six hours after the last dose, he underwent a pylorus-preserving pancreatoduodenectomy. Because of the presence of high-grade dysplasia detected by intraoperative histological examination of a distal section, a spleen preserving total pancreatectomy was performed. The postoperative course was uneventful. The patient died 32 months after surgery because of local recurrence. Histopathology showed G3 pancreatic ductal adenocarcinoma infiltrating the anterior and posterior peripancreatic tissue, duodenal wall and intrapancreatic common bile duct, with sarcoma-like foci and a component of intraductal tumor involving the common bile duct. In the distal pancreas, widespread foci of pancreatic intraepithelial neoplasia (PanI2-3) were found. The Ki-67 proliferation index was 16%. TNM staging was pT3 pN1 R1. Sections were immunostained for the T-lymphocyte marker CD3 and for the dendritic cell marker CD1a. Intratumoral infiltration was high for CD1a+ cells and mild for CD3+ cells. Preoperative immunotherapy with interleukin-2 may contribute to massive stromal infiltration of immune cells in pancreatic adenocarcinoma. This may prolong the survival even in the presence of negative prognostic factors (age >65 years, tumor diameter >20 mm, R1, tumor grade G3). PMID:18705415

  19. In vivo distribution of recombinant interleukin-2-activated autologous lymphocytes administered by intra-arterial infusion in patients with renal cell carcinoma

    SciTech Connect

    Morita, T.; Yonese, Y.; Minato, N.

    1987-03-01

    Recombinant interleukin-2 (RIL 2)-activated autologous peripheral blood lymphocytes (PBL) were infused directly into the renal arteries of 3 patients with renal cell carcinoma, and the in vivo distribution of the infused cells was investigated. In vitro studies to define the optimal culture conditions indicated that maximal lymphokine-activated killer activity was observed at around 10-20 days in culture, as judged by the cytotoxicity against fresh allogenic tumor cells. Maximal expression of the interleukin-2 receptor was also obtained at around 10 days. PBL collected by leukopheresis from each patient were thus cultured for 10 days with RIL 2, labeled with /sup 111/In-oxine, and then infused directly into the renal artery of the affected kidney via a catheter. Radioactivity in the infused side of the kidneys increased immediately after the infusion but then gradually decreased. Radioactivity in the lungs also rapidly increased within the first hour but then cleared gradually, whereas that in the liver and spleen tended to increase steadily. Nevertheless, at 48 hours, the infused side of the kidneys retained levels of radioactivity comparable to those seen in the liver and spleen, while the levels seen in the lungs were already close to background levels. The radioactivity in the areas corresponding to tumors remained consistently higher than that in the normal parts of the affected kidneys. The direct comparison of the radioactivity distribution pattern with the macroscopic appearance of surgically resected kidneys indicated that the accumulation of radioactivity was indeed selectively associated with the tumor tissues in the kidneys, except for a case in which the tumor was quite necrotic and hypovascular.

  20. Evaluation of Tumour Necrosis Factor Alpha, Interleukin-2 Soluble Receptor, Nitric Oxide Metabolites, and Lipids as Inflammatory Markers in Type 2 Diabetes Mellitus

    PubMed Central

    Pereira, Flvia Ozorio; Frode, Tnia Silvia; Medeiros, Yara Santos

    2006-01-01

    This study compared the results of tumour necrosis factor alpha (TNF-?), interleukin-2 soluble receptor (sIL-2R), nitric oxide metabolites (NOx), C-reactive protein (CRP), and lipids (total cholesterol, high-density lipoprotein (HDL-cholesterol), lowdensity lipoprotein (LDL-cholesterol), and triglycerides) between control group (nondiabetic subjects) and overweight type 2 DM subjects. To restrict the influence of variables that could interfere in the interpretation of data, subjects with obesity and/or acute or chronic inflammatory disease, haemoglobinopathies, recent use of antibiotics, antiinflammatory drugs, and trauma were excluded. Type 2 DM patients (n = 39; age 53.3 9.0 years; median glycated haemoglobin A1c < 8%) presented higher levels of TNF-?, triglycerides (P < .01), NOx and sIL-2R (P < .05) than control group (n = 28; age 39.7 14.1 years). CRP, LDL-cholesterol, total cholesterol, and HDL-cholesterol did not differ among groups. Diabetic women (n = 21) had higher levels of TNF-?, total cholesterol, LDL-cholesterol, and HDL-cholesterol than diabetic men (n = 18) (P < .05), but there were no differences among sexes in the control group. This study indicates that increased level of proinflammatory markers occurs in type 2 DM even in the absence of obesity and marked hyperglycaemia, confirming that the inflammation course of the atherosclerotic process is more severe in diabetic patients than in nondiabetic subjects. PMID:16864902

  1. The pathogenesis of experimental toxic shock syndrome: the role of interleukin-2 in the induction of hypotension and release of cytokines.

    PubMed

    Tokman, M G; Carey, K D; Quimby, F W

    1995-02-01

    Toxic shock syndrome (TSS) is a multisystem disorder characterized by fever, hypotension, and involvement of three other organ systems. The etiologic agent is a toxigenic strain of Staphylococcus aureus which secretes the exotoxin, TSST-1. The toxin is a superantigen which stimulates the immune system to produce interleukin-1 (IL-1), interleukin-2, and tumor necrosis factor (TNF). We hypothesized that TSST-1 induces the release of IL-2 which in turn is either directly involved or acts via an additional mediator to produce hypotension. We submitted four pairs of normal anesthetized adult female baboons to intravenous boluses of TSST-1. One baboon in each pair received anti-IL-2 intravenously and anti-IL-2 receptor intrathyroidally 15 min prior to TSST-1. The other baboon received the same dose and placement of anti-sheep red blood cell antibody. Systolic and diastolic blood pressure was recorded continuously and mean arterial pressure was calculated and plotted. IL-1, IL-2, IL-6, and TNF were measured in serum at varying times before and after toxin administration. Systolic, diastolic, and mean arterial pressure were significantly lower in the sham-treated group versus the experimental (anti-IL-2/IL-2R) group (p < .05 for all variables). In addition no differences were seen in any of the measurements between experimentally treated baboons and those receiving no TSST-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7749941

  2. Interferon-gamma (IFN-gamma) and interleukin-2 in the generation of lymphokine-activated killer cell cytotoxicity--IFN-gamma-induced suppressive activity.

    PubMed

    Toledano, M; Mathiot, C; Michon, J; Andreu, G; Lando, D; Brandely, M; Fridman, W H

    1989-01-01

    Incubation of human lymphocytes with recombinant interleukin-2 (rIL-2) results in the generation of lymphokine-activated killer (LAK) cells capable of lysing a wide variety of tumor cells. The present study was undertaken to examine the effect of recombinant gamma interferon (rIFN-gamma) on LAK cell cytotoxicity generated from different peripheral blood mononuclear cell (PBMC) subpopulations. When unseparated PBMC were stimulated by rIL-2 and rIFN-gamma, the latter induced a transient enhancement after 2 days followed by a suppression of LAK cell cytotoxicity at day 6. Enhancement of LAK cell cytotoxicity was moderate and inconstant, whereas the inhibition was strong and observed with all the donors tested. This suppression was not associated with a decrease in the [3H]thymidine uptake. PBMC depleted of adherent cells were more sensitive to the stimulation by rIL-2 and the induced cytotoxicity was not modified by rIFN-gamma. Monocyte-enriched plastic-adherent cells, when incubated with rIL-2 and rIFN-gamma, became cytotoxic after 2-3 days of culture and inhibited LAK cell activity after 5-6 days. Collectively, our results suggest that rIFN-gamma affects LAK cell cytotoxicity through the activation of plastic-adherent, monocyte-rich, cells which modulate natural killer cells, first in a positive, then in a negative way. PMID:2513112

  3. Natural Variation in Interleukin-2 Sensitivity Influences Regulatory T-Cell Frequency and Function in Individuals With Long-standing Type 1 Diabetes.

    PubMed

    Yang, Jennie H M; Cutler, Antony J; Ferreira, Ricardo C; Reading, James L; Cooper, Nicholas J; Wallace, Chris; Clarke, Pamela; Smyth, Deborah J; Boyce, Christopher S; Gao, Guo-Jian; Todd, John A; Wicker, Linda S; Tree, Timothy I M

    2015-11-01

    Defective immune homeostasis in the balance between FOXP3(+) regulatory T cells (Tregs) and effector T cells is a likely contributing factor in the loss of self-tolerance observed in type 1 diabetes (T1D). Given the importance of interleukin-2 (IL-2) signaling in the generation and function of Tregs, observations that polymorphisms in genes in the IL-2 pathway associate with T1D and that some individuals with T1D exhibit reduced IL-2 signaling indicate that impairment of this pathway may play a role in Treg dysfunction and the pathogenesis of T1D. Here, we have examined IL-2 sensitivity in CD4(+) T-cell subsets in 70 individuals with long-standing T1D, allowing us to investigate the effect of low IL-2 sensitivity on Treg frequency and function. IL-2 responsiveness, measured by STAT5a phosphorylation, was a very stable phenotype within individuals but exhibited considerable interindividual variation and was influenced by T1D-associated PTPN2 gene polymorphisms. Tregs from individuals with lower IL-2 signaling were reduced in frequency, were less able to maintain expression of FOXP3 under limiting concentrations of IL-2, and displayed reduced suppressor function. These results suggest that reduced IL-2 signaling may be used to identify patients with the highest Treg dysfunction and who may benefit most from IL-2 immunotherapy. PMID:26224887

  4. Interleukin-2 (IL-2) Regulates the Accessibility of the IL-2-Responsive Enhancer in the IL-2 Receptor α Gene to Transcription Factors

    PubMed Central

    Rusterholz, Corinne; Henrioud, Patricia Corthésy; Nabholz, Markus

    1999-01-01

    Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) α subunit by antigen and by IL-2 itself. IL-2 induces IL-2Rα transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2Rα expression. In cells induced to transiently express IL-2Rα with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2Rα transcription by making IL-2Rα chromatin accessible to transcription factors. PMID:10082534

  5. Phase IB study of low-dose intraperitoneal recombinant interleukin-2 in patients with refractory advanced ovarian cancer: rationale and preliminary report.

    PubMed

    Beller, U; Chachoua, A; Speyer, J L; Sorich, J; Dugan, M; Liebes, L; Hayes, R; Beckman, E M

    1989-09-01

    The biological activity of recombinant Interleukin-2 (rIL-2) administered intraperitoneally (ip) has not been determined and may differ significantly from the maximum tolerated dose (MTD). In this trial, the pharmacokinetics, toxicity, and biologic activity of a single ip dose were studied initially followed a week later by a 5-day ip rIL-2 given for 2 weeks every 28 days. Planned dose escalation was from 2 x 10(3) to 2 x 10(7) U given in 2 liters of D5W. Drug was obtained from the NCI and was administered through an ip port. Four patients received 1 U/ml and four patients received 10 U/ml. Preliminary data demonstrate an increase in the peritoneal fluid mononuclear cell count. Mononuclear cell phenotyping tested in the first eight patients showed a modest increase in Leu 2a+, Leu 15- cells, corresponding to CTL. A similar increase in Leu 19+ cells was also demonstrated (NK cells). Soluble IL-2 receptor was elevated in peritoneal fluid. Cytotoxicity against K562 and Daudi cell lines was not observed at the first two dose levels. Toxicity of treatment was minimal and related to abdominal distention. No objective responses were seen but in one patient we documented a reduction in serum CA-125 levels. The observed biologic response and lack of toxicity is promising and justifies further exploration of this immune-modulating approach. PMID:2788602

  6. Effect of interleukin 2 on urinary excretion of degradation products of prostacyclin and thromboxane A2 in patients with ovarian cancer.

    PubMed Central

    Aitokallio-Tallberg, A.; Lehtovirta, P.; Vartiainen, J.; Ylikorkala, O.

    1995-01-01

    We studied the effect of intraperitoneal recombinant interleukin 2 (rIL-2) on the production of prostacyclin (PGI2) and thromboxane A2 (TxA2) in six patients with metastatic ovarian malignancy. Time-span urine samples collected before and after 17 intraperitoneal instillations of IL-2 (6 x 10(5) IU m-2) were assessed for 2,3-dinor-6-keto-prostaglandin F1 alpha (dinor-6-keto; a metabolite reflecting the in vivo product of PGI2) and 2,3-dinor-thromboxane B2 (dinor-TxB2; a metabolite reflecting the production of TxA2). Analysis was by high-pressure liquid chromatography, followed by radioimmunoassay. Recombinant IL-2 administration was accompanied by a significant rise (85%; P < 0.02) in the output of dinor-6-keto within the first 2 h, and this elevation persisted for up to 6 h. Moreover, output of dinor-TxB2 also rose; this rise (30%) was significant (P < 0.02) 6 h after the instillation. These effects may, in some yet unknown manner, prove significant in the anti-cancer action of rIL-2. PMID:7547215

  7. Synergistic induction by calcium ionophore and phorbol ester of interleukin-2 (IL-2) receptor expression, IL-2 production, and proliferation in autoimmune MRL/MP-lpr mice.

    PubMed Central

    Koizumi, T; Nakao, Y; Matsui, T; Katakami, Y; Nakagawa, T; Fujita, T

    1986-01-01

    MRL/MP-lpr/lpr (MRL/l) mice spontaneously develop an age-related autoimmune disease concomitant with interleukin-2 (IL-2) defects. Induction of IL-2 receptor (IL-2R), IL-2 production and subsequent de novo DNA synthesis in MRL/l mice by the tumour-promoting phorbol ester 12-o-tetradecanoyl phorbol 13-acetate (TPA) and calcium ionophore (A23187) were examined. These two compounds given together induced significant IL-2R expression, IL-2 production, and de novo DNA synthesis of spleen cells from this murine strain, as did concanavalin A (Con A) plus TPA. TPA and A23187 may bypass the early steps of activation by mitogens in murine lymphocytes. However, even though these IL-2 defects could be overcome to some extent, the response of MRL/l mice to these stimuli was considerably lower than the enhanced IL-2R expression and IL-2 production of MRL/MP-+/+(MRL/n) control mice. These results suggested that the failure to respond to mitogens in these mice may be due, at least in part, to failure of receptor signal transduction, and to defects of molecular and biochemical reactions following signal transduction. PMID:3093371

  8. Immunogenicity of oral vaccination with Lactococcus lactis derived vaccine candidate antigen (UreB) of Helicobacter pylori fused with the human interleukin 2 as adjuvant.

    PubMed

    Zhang, Hong-xin; Qiu, Yu-yu; Zhao, Ying-hui; Liu, Xin-ting; Liu, Ming; Yu, Ai-lian

    2014-02-01

    Helicobacter pylori (H. pylori) infection remains a significant global public health problem. Vaccine, especially edible vaccine, is considered to be effective in the management of H. pylori infections. By using recombinant technology, Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. The aim of this study was to produce edible UreB (urease B) vaccine derived from L. lactis against H. pylori. The UreB subunit is the most effective and common immunogen of all strains of H. pylori. The UreB was produced as a chimeric protein fused with IL-2 (human interleukin 2) as the mucosal adjuvant. Mucosal immunization of mice with recombinant L. lactis NZ9000 containing the UreB-IL-2 protein elicited more anti-UreB antibody that specifically bounded to the purified bacterial UreB protein and more cytokines such as IFN-?, IL-4, and IL-17, and had a lower H. pylori burden and urease activity than control mice. These results suggest that the recombinant L. lactis expressing UreB-IL-2 can be potentially used as an edible vaccine for controlling H. pylori infection. PMID:24036137

  9. Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo

    PubMed Central

    YOU, QI; YAO, YUAN; ZHANG, YUANLONG; FU, SONGBIN; DU, MEI; ZHANG, GUANGMEI

    2015-01-01

    The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein-human interleukin-2 (pEGFP-hIL-2) was formed. The plasmid was stably transfected into the AF-MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL-2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF-MSCs exhibited high motility during migration in vivo, and the vector, pEGFP-hIL-2 can be stably transfected into AF-MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors. PMID:26179662

  10. Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2.

    PubMed

    Hamdi, Safouane M; Cariven, Clotilde; Coronas, Sophie; Malet, Nicole; Chap, Hugues; Perret, Bertrand; Salles, Jean-Pierre; Record, Michel

    2008-05-01

    Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders. PMID:18423386

  11. A pilot study to evaluate the effects of C1 esterase inhibitor on the toxicity of high-dose interleukin 2.

    PubMed Central

    Ogilvie, A. C.; Baars, J. W.; Eerenberg, A. J.; Hack, C. E.; Pinedo, H. M.; Thijs, L. G.; Wagstaff, J.

    1994-01-01

    In a pilot study six patients received 4 days' treatment with interleukin 2 (IL-2) [cumulative dose (CD) 264 +/- 26 x 10(6) IU m-2] and C1 esterase inhibitor (C1-INH) (loading dose 2,000 U, followed by 500-1,000 U twice daily). Toxicity was compared with that in patients given 4 days' treatment with standard (CD 66 +/- 12 x 10(6) IU m-2) or escalating-dose (CD 99 +/- 8 x 10(6) IU m-2) IL-2. IL-2-induced hypotension was equivalent and complement activation was less after IL-2 + C1-INH (C3a = 10.5 +/- 3.2 nmol l-1) than following standard (14.1 +/- 8.4 nmol l-1) or escalating-dose (18.3 +/- 2.9 nmol l-1) IL-2. This study demonstrates that C1-INH administration during IL-2 treatment is safe and warrants further study to evaluate its ability to ameliorate IL-2-induced toxicity. PMID:8123494

  12. Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo.

    PubMed

    You, Qi; Yao, Yuan; Zhang, Yuanlong; Fu, Songbin; Du, Mei; Zhang, Guangmei

    2015-10-01

    The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein?human interleukin?2 (pEGFP?hIL?2) was formed. The plasmid was stably transfected into the AF?MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL?2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF?MSCs exhibited high motility during migration in vivo, and the vector, pEGFP?hIL?2 can be stably transfected into AF?MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors. PMID:26179662

  13. Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12.

    PubMed Central

    MacDonald, H L; Neway, J O

    1990-01-01

    We examined the ability of transformed Escherichia coli cells in fermentor cultures to accumulate interleukin-2 (IL-2) intracellularly under temperature-regulated control of the phage lambda pL promoter. Induction of expression was undertaken at different culture optical densities, and specific IL-2 accumulation was found to decrease with increasing cell density at induction. Induction at higher culture optical densities was also accompanied by decreased growth during induction and increased acetate accumulation in the culture medium. Experiments were undertaken to study the effect of replacing spent medium by perfusion with fresh medium both before induction and during IL-2 expression at high cell density. Improved IL-2 expression was seen only when perfusion was continued past 1.6 h after the start of induction, and it was accompanied by a significant reduction in acetate buildup. Further improvements were not seen when perfusion was continued beyond hour 3 of induction. Replenishing medium components and decreasing the concentration of diffusible inhibitors before induction did not alleviate acetate buildup, growth limitation, or limitation of IL-2 synthesis. These results suggested that accumulation of diffusible inhibitors such as acetate during induction may be a significant factor limiting IL-2 expression in high-density cultures, but other factors intrinsic to the organism or the protein also played a major role. PMID:2180368

  14. 65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites

    SciTech Connect

    Zu, Youli; Shigesada, Katsuya; Hanaoka, Masao; Namba, Yuziro ); Nishida, Eisuke ); Kubota, Ichiro ); Kohno, Michiaki )

    1990-09-11

    The authors have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin. In this paper, they present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.

  15. A cis element required for induction of the interleukin 2 enhancer by human T-cell leukemia virus type I binds a novel Tax-inducible nuclear protein.

    PubMed Central

    Li, M; Siekevitz, M

    1993-01-01

    The 40-kDa nuclear protein Tax encoded by human T-cell leukemia virus type I (HTLV-I) can transcriptionally activate the interleukin 2 (IL-2) enhancer even in the presence of the immunosuppressant cyclosporin A, which inhibits the activation of the IL-2 enhancer by T-cell mitogens. We have identified a Tax-responsive element (TxRE) from -164 to -145 bp in the IL-2 enhancer which is sufficient to confer Tax responsiveness. A 45-kDa nuclear protein (TxRE-binding factor [TxREF]), present in Tax-expressing Jurkat cell lines but not in Jurkat cells without Tax, specifically interacts with the 5' TxRE sequence from -164 to -154. Deletion or mutation of this 5' TxRE sequence removes the binding of TxREF in vitro and dramatically reduces Tax activity in vivo. In addition, this site is responsible for the cyclosporin A-resistant expression of the IL-2 enhancer in the presence of Tax. Although the TxREF binding site contains an NF-kappa B like motif, UV cross-linking studies as well as gel retardation analysis reveal that TxREF is distinct from NF-kappa B. These results demonstrate that TxREF is a novel Tax-inducible DNA-binding protein and that TxRE plays a crucial role in mediating Tax-induced IL-2 gene expression. Images PMID:8413248

  16. The shared and contrasting roles of interleukin-2 (IL-2) and IL-15 in the life and death of normal and neoplastic lymphocytes: implications for cancer therapy

    PubMed Central

    Waldmann, Thomas A.

    2015-01-01

    Interleukin-2 (IL2) and IL15, members of the 4?-helix bundle family of cytokines, play pivotal roles in the control of the life and death of lymphocytes. Although their heterotrimeric receptors have two receptor subunits in common these two cytokines have contrasting roles in adaptive immune responses. The unique role of IL2 through maintenance of fitness of regulatory T cells (Treg) and activation-induced cell death (AICD) is the elimination of self-reactive T cells to prevent autoimmunity. In contrast to IL2, IL15 is dedicated to the prolonged maintenance of memory T-cell responses to invading pathogens. Blockade of IL2 and IL15 using monoclonal antibodies has been reported to be of value in the treatment of patients with leukemia, autoimmune disorders and in the prevention of allograft rejection. IL2 has been approved by the FDA for the treatment of patients with malignant renal cell cancer and metastatic malignant melanoma. Clinical trials involving recombinant human IL15 given by bolus infusions have been completed, and by subcutaneous and continuous intravenous infusions are underway in patients with metastatic malignancy. Furthermore, clinical trials are being initiated that employ the combination of IL15 with IL15R?+/? IgFc. PMID:25736261

  17. Direct inhibition of interleukin-2 receptor alpha-mediated signaling pathway induces G1 arrest and apoptosis in human head-and-neck cancer cells.

    PubMed

    Kuhn, Deborah J; Dou, Q Ping

    2005-05-15

    Overexpression of the interleukin-2 receptor (IL-2R) alpha chain in tumor cells is associated with tumor progression and a poor patient prognosis. IL-2Ralpha is responsible for the high affinity binding of the receptor to IL-2, leading to activation of several proliferative and anti-apoptotic intracellular signaling pathways. We have previously shown that human squamous cell carcinoma of a head-and-neck line (PCI-13) genetically engineered to overexpress IL-2Ralpha exhibit increased transforming activity, proliferation, and drug resistance, compared to the vector control cells (J Cell Biochem 2003;89:824-836). In this study, we report that IL-2Ralpha(+) cells express high levels of total and phosphorylated Jak3 protein and are more resistant to apoptosis induced by a Jak3 inhibitor than the control LacZ cells. Furthermore, we used daclizumab, a monoclonal antibody specific to IL-2Ralpha, and determined the effects of IL-2Ralpha inhibition on cell cycle and apoptosis as well as the involvement of potential cell cycle and apoptosis regulatory proteins. We found that daclizumab induces G(1) arrest, associated with down-regulation of cyclin A protein, preferentially in IL-2Ralpha(+) cells, but not in LacZ cells. In addition, daclizumab activates apoptotic death program via Bcl-2 down-regulation preferentially in IL-2Ralpha(+) cells. Finally, daclizumab also sensitizes IL-2Ralpha(+) cells to other apoptotic stimuli, although the effect is moderate. These results indicate that daclizumab inhibits the proliferative potential of IL-2Ralpha(+) cells via inhibition of cell cycle progression and induction of apoptosis. PMID:15779002

  18. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes

    SciTech Connect

    Grimm, E.A.; Mazumder, A.; Zhang, H.Z.; Rosenberg, S.A.

    1982-06-01

    Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

  19. Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites

    PubMed Central

    Li, Zhuoran; Tang, Xinming; Suo, Jingxia; Qin, Mei; Yin, Guangwen; Liu, Xianyong; Suo, Xun

    2015-01-01

    Chicken coccidiosis, caused by Eimeria sp., occurs in almost all poultry farms and causes huge economic losses in the poultry industry. Although this disease could be controlled by vaccination, the reduced feed conservation ratio limits the widespread application of anticoccidial vaccines in broilers because some intermediate and/or low immunogenic Eimeria sp. only elicit partial protection. It is of importance to enhance the immunogenicity of these Eimeria sp. by adjuvants for more effective prevention of coccidiosis. Cytokines have remarkable effects on the immunogenicity of antigens. Interleukin 2 (IL-2), for example, significantly stimulates the activation of CD8+ T cells and other immune cells. In this study, we constructed a transgenic Eimeria mitis line (EmiChIL-2) expressing chicken IL-2 (ChIL-2) to investigate the adjuvant effect of ChIL-2 to enhance the immunogenicity of E. mitis against its infection. Stable transfected EmiChIL-2 population was obtained by pyrimethamine selection and verified by PCR, genome walking, western blotting and indirect immunofluorescence assay. Cellular immune response, E. mitis-specific IFN-γ secretion lymphocytes in the peripheral blood mononuclear cells, stimulated by EmiChIL-2 was analyzed by enzyme-linked immunospot assay (ELISPOT). The results showed that EmiChIL-2 stimulated a higher cellular immune response compared with that of the wild-type parasite infection in chickens. Moreover, after the immunization with EmiChIL-2, elevated cellular immune response as well as reduced oocyst output were observed These results indicated that ChIL-2 expressed by Eimeria sp. functions as adjuvant and IL-2 expressing Eimeria parasites are valuable vaccine strains against coccidiosis. PMID:26082759

  20. Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694.

    PubMed

    Zhong, Yiming; Dong, Shuai; Strattan, Ethan; Ren, Li; Butchar, Jonathan P; Thornton, Kelsey; Mishra, Anjali; Porcu, Pierluigi; Bradshaw, J Michael; Bisconte, Angelina; Owens, Timothy D; Verner, Erik; Brameld, Ken A; Funk, Jens Oliver; Hill, Ronald J; Johnson, Amy J; Dubovsky, Jason A

    2015-03-01

    Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases. PMID:25593320

  1. Downregulation of JAK3 protein levels in T lymphocytes by prostaglandin E2 and other cyclic adenosine monophosphate-elevating agents: impact on interleukin-2 receptor signaling pathway.

    PubMed

    Kolenko, V; Rayman, P; Roy, B; Cathcart, M K; O'Shea, J; Tubbs, R; Rybicki, L; Bukowski, R; Finke, J

    1999-04-01

    The Janus kinase, JAK3 plays an important role in interleukin-2 (IL-2)-dependent signal transduction and proliferation of T lymphocytes. Our findings show that prostaglandin E2 (PGE2) can inhibit upregulation of JAK3 protein in naive T cells and can downregulate its expression in primed cells. Reduction in JAK3 was selective because expression of other tyrosine kinases (JAK1, p56(lck), and p59(fyn)) and signal transducer and activator of transcription (STAT)5, which are linked to IL-2 receptor (IL-2R) signaling pathway, were not affected. Inhibition of JAK3 may be controlled by intracellular cyclic adenosine monophosphate (cAMP) levels, as forskolin, a direct activator of adenylate cyclase and dibutyryl cAMP (dbcAMP), a membrane permeable analogue of cAMP suppressed JAK3 expression. Moreover, 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase, potentiated PGE2-induced suppression of JAK3. In naive T cells, but not primed T cells, PGE2 and other cAMP elevating agents also caused a modest reduction in surface expression of the common gamma chain (gammac) that associates with JAK3. The absence of JAK3, but not IL-2R in T cells correlated with impaired IL-2-dependent signal transduction and proliferation. The alteration in IL-2 signaling included decreased tyrosine phosphorylation and DNA binding activity of STAT5 and poor induction of the c-Myc and c-Jun pathways. In contrast, IL-2-dependent induction of Bcl-2 was unaffected. These findings suggest that suppression of JAK3 levels may represent one mechanism by which PGE2 and other cAMP elevating agents can inhibit T-cell proliferation. PMID:10090941

  2. Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression.

    PubMed Central

    Nakajima, F; Khanna, A; Xu, G; Lagman, M; Haschemeyer, R; Mouradian, J; Wang, J C; Stenzel, K H; Rubin, A L; Suthanthiran, M

    1994-01-01

    Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans. Images PMID:8058730

  3. Induction of interleukin-2 transcription by the hamster polyomavirus middle T antigen: a role for Fyn in T cell signal transduction.

    PubMed

    Brizuela, L; Ulug, E T; Jones, M A; Courtneidge, S A

    1995-02-01

    The transforming protein of mouse polyomavirus, the mouse middle T antigen (MomT), and its counterpart in the hamster polyomavirus, the hamster middle T antigen (HamT), interact with a number of cellular proteins. Among these are members of the Src family of tyrosine kinases, the phosphatidylinositol 3-kinase, the serine/threonine phosphatase PP2A and the adaptor protein Shc (in the case of MomT). However, both the relative affinity of these antigens for the members of the Src family and the tumor profile induced by their respective viruses are quite distinct. Particularly noteworthy are the preferential binding of Fyn by HamT and the induction of lymphoid malignancies by the hamster polyomavirus. Here we report that, when expressed in fibroblasts, HamT also associated with phospholipase C gamma (PLC gamma), which led to an increased intracellular concentration of inositol-1, 4, 5-trisphosphate. We also show that expression of HamT in the mouse T cell line EL4 was sufficient to induce transcription from interleukin-2 (IL-2), NFAT and NF kappa B reporter constructs. The immunosuppressant FK506 as well as dominant negative alleles of Ras and Raf inhibited HamT-induced IL-2 transcription. This, together with the observation of NFAT responses, suggests that the action of HamT depended at least in part on the integrity of signal transduction pathways elicited by activated PLC gamma. Furthermore, dominant negative Fyn but not the equivalent allele of Lck blocked HamT activation of IL-2 transcription, while both Lck and Fyn dominant negative alleles blocked LT cell receptor-mediated IL-2 transcriptional activation. These results support the hypothesis that Fyn is involved in signal transduction events leading to IL-2 transcriptional activation in T cells. Finally, the activation of IL-2 transcription by HamT and not by MomT shown here parallels the ability of the hamster polyomavirus to induce lymphoid malignancies. PMID:7875200

  4. Influence of Foreign DNA Introduction and Periplasmic Expression of Recombinant Human Interleukin-2 on Hydrogen Peroxide Quantity and Catalase Activity in Escherichia coli

    PubMed Central

    Mahmoudi Azar, Lena; Mehdizadeh Aghdam, Elnaz; Karimi, Farrokh; Haghshenas, Babak; Barzegari, Abolfazl; Yaghmaei, Parichehr; Hejazi, Mohammad Saeid

    2013-01-01

    Purpose: Oxidative stress is generated through imbalance between composing and decomposing of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Effect of cytoplasmic recombinant protein expression on Hydrogen peroxide concentration and catalase activity was previously reported. In comparison with cytoplasm, periplasmic space has different oxidative environment. Therefore, in present study we describe the effect of periplasmic expression of recombinant human interleukin-2 (hIL-2) on H2O2 concentration and catalase activity in Escherichia coli and their correlation with cell growth. Methods: Having constructed pET2hIL2 vector, periplasmic expression of hIL-2 was confirmed. Then, H2O2 concentration and catalase activity were determined at various ODs. Wild type and empty vector transformed cells were used as negative controls. Results: It was shown that H2O2 concentration in hIL-2 expressing cells was significantly higher than its concentration in wild type and empty vector transformed cells. Catalase activity and growth rate reduced significantly in hIL-2 expressing cells compared to empty vector transformed and wild type cells. Variation of H2O2 concentration and catalase activity is intensive in periplasmic hIL-2 expressing cells than empty vector containing cells. Correlation between H2O2 concentration elevation and catalase activity reduction with cell growth depletion are also demonstrated. Conclusion: Periplasmic expression of recombinant hIL-2 elevates the host cells hydrogen peroxide concentration possibly due to reduced catalase activity which has consequent suppressive effect on growth rate. PMID:24312866

  5. Unique pattern of interleukin 2 receptor expression by lymphocytes in response to anti-LEU 4: relationship to monocyte accessory cell function

    SciTech Connect

    Prince, H.E.; John, J.K.

    1986-03-05

    The relationship between early interleukin 2 receptor (IL2R) expression and subsequent DNA synthesis by mitogen-stimulated human mononuclear cells (MC) was studied. For serial dilutions of a given mitogen, the percentage of lymphocytes expressing IL2R after 1 day of culture was plotted vs /sup 3/H-thymidine incorporation on day 3, and the IL2R value associated with 50,000 counts per minute (IL2R-50K) determined. A mean IL2R-50K value of 7 characterized PHA, Con A, and OKT3 responses, while a higher value of 29 characterized anti-Leu 4 (L4) responses. As tested on OKT3 and L4 systems, exogenous IL2 did not alter IL2R-50K values. Because OKT3 and L4 both recognize the lymphocyte CD3 antigen, but react with different monocyte Fc receptors, the role of monocytes in producing elevated L4 IL2R-50K values was explored. MC from healthy L4 nonresponders (NR), induced to proliferate by L4 in the presence of responder (R) monocytes, also yielded an elevated mean IL2R-50K value of 31. In contrast, direct stimulation of R-MC, NR-MC, or NR-MC plus R monocytes by L4-coated sepharose beads produced mean IL2R-50K values of 12 or 13. These findings suggest that crosslinking of lymphocyte-bound soluble L4 by R monocytes leads to uniquely elevated pattern of lymphocyte IL2R expression. Crosslinking mediated by L4-beads, however, leads to a more conventional pattern of IL2R expression, even though R monocytes may be present.

  6. Immunotherapy of murine sarcomas using lymphokine activated killer cells: optimization of the schedule and route of administration of recombinant interleukin-2

    SciTech Connect

    Ettinghausen, S.E.; Rosenberg, S.A.

    1986-06-01

    Interleukin-2 (IL-2) at high doses or at low doses in concert with lymphokine-activated killer (LAK) cells can produce regression of established pulmonary and hepatic metastases from a variety of tumors in mice. IL-2 appears to mediate its antitumor effect through the generation of LAK cells in vivo from endogenous lymphocytes and by the stimulation of host and transferred LAK cell proliferation in tissues. In this paper we have investigated different strategies for IL-2 administration to determine which regimen produced maximal in vivo proliferation and optimal immunotherapeutic efficacy of LAK cells. Tissue expansion of lymphoid cells was assessed using an assay of in vivo labeling of dividing cells by the thymidine analogue, 5-(/sup 125/I)iododeoxyuridine. The therapeutic effect of the different IL-2 administration protocols was determined by evaluating their efficacy in the treatment of established, 3-day pulmonary metastases from sarcomas in mice. The selection of IL-2 injection regimens for evaluation was based upon pharmacokinetic studies of IL-2 in mice. A single i.v. or i.p. dose yielded high peak IL-2 levels that could be measured for only a few hours after injection, while IL-2 given i.p. thrice daily produced titers that were detectable throughout the study periods (greater than or equal to 6 units/ml of serum after 100,000 units of IL-2 i.p. thrice daily). Using the proliferation and therapy models, we tested the same cumulative daily doses of IL-2 administered by i.v. or i.p. once daily, or i.p. thrice daily regimens. The i.p. thrice daily protocol stimulated greater lymphoid cell proliferation in the lungs, for example, than did the other regimens.

  7. Clinical and immune modulatory effects of alternative weekly interleukin-2 and interferon alfa-2a in patients with advanced renal cell carcinoma and melanoma.

    PubMed Central

    Pichert, G.; Jost, L. M.; Fierz, W.; Stahel, R. A.

    1991-01-01

    The clinical and immune modulatory effects of interleukin-2 (IL-2) and interferon (INF) alfa-2a were examined in a phase II study in patients with metastatic renal cell carcinoma (six patients) and melanoma (eight patients). Treatment consisted in IL-2 3 MU/m2 continuous infusion days 1-4 and INF alfa-2a 6 MU/m2 subcutaneously day 1 and 4, both given on alternate weeks. Tumour response was assessed after four cycles of treatment or earlier, if necessary. Patients with stable disease or response were to be continued for another nine cycles or up to disease progression. The 14 patients received a total of 60 cycles of treatment. Major toxicities (WHO Grade III/IV) were fever, capillary leak syndrome with hypotension, nausea and vomiting, erythema with pruritus, leuco- and thrombopenia and sepsis with staphylococcus aureus. Five of 14 patients (36%) developed a self limiting autoimmune thyroiditis with HLA-DR expression on thyrocytes. Long term treatment toxicity was moderate with an average weight loss of 5% and an average fall in Karnofsky index of 10% compared to baseline. No responses were seen in renal cell carcinoma, two patients with melanoma had a partial and two a minor response with a duration of 1-7 months. Serial measurements of immune modulatory parameters showed a functional response to treatment with an increase of NK- and LAK-activity during the first two cycles, followed by a plateau and decrease during the third and fourth cycles. These findings were paralleled by a successive decline in treatment induced INF gamma response. These findings suggest, that alternative weekly treatment with IL-2 and INF alfa-2a results in an exhaustion of lytic capacity of NK- and LAK-cells and an attenuation of secondary cytokine release. Images Figure 2 PMID:1997108

  8. Mechanism of action of interleukin-2 (IL-2)-Bax, an apoptosis-inducing chimaeric protein targeted against cells expressing the IL-2 receptor.

    PubMed Central

    Aqeilan, Rami; Kedar, Rotem; Ben-Yehudah, Ahmi; Lorberboum-Galski, Haya

    2003-01-01

    The chimaeric protein interleukin-2 (IL-2)-Bax was designed to target and kill specific cell populations expressing the IL-2 receptor. However, it is not well understood how IL-2-Bax causes target cells to die. In the present study, we investigated the pathway of apoptosis evoked by IL-2-Bax and the possible involvement of endogenous Bax in this process. We report here that, upon internalization of IL-2-Bax into target cells, it is localized first mainly in the nucleus, and only later is it translocated to the mitochondria. Similarly, endogenous Bax is also partially localized in the nucleus, and accumulates mainly in this compartment soon after physiological triggering of apoptosis. Despite the fact that Bax has no nuclear localization sequence, our data suggest that Bax has one or more physiological roles and/or substrates within the nucleus. Indeed, a dramatic repression of nuclear Tax protein expression was induced following treatment of HUT-102 cells with IL-2-Bax, similar to what occurs following serum deprivation of these cells. Unexpectedly, induction of apoptosis using IL-2-Bax was preceded by enhanced expression of newly synthesized Bax protein and suppression of Bcl-2. This imbalance between the pro- and anti-apoptotic genes was associated with p53 induction, although IL-2-Bax activity was also evident in cells lacking p53 expression. By studying the mechanism of action of IL-2-Bax, we were able to follow the intrinsic events and their cascade that culminates in cell death. We have shown that the ability of IL-2-Bax to affect the intracellular apoptotic machinery within the target cells, and to cause the cells to die, uses a mechanism similar to that induced following a normal apoptotic signal. PMID:12405905

  9. Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells.

    PubMed

    Rao, Y P; Buckley, D J; Buckley, A R

    1995-10-01

    Several serine/threonine and tyrosine kinase signal transduction pathways have been recently linked to prolactin (PRL) action in lymphoid cells. Utilizing the lactogen-dependent, rat pre-T lymphoma cell line, Nb2-11, and the autonomous subline, Nb2-SFJCD1, studies were conducted to determine whether PRL- or interleukin-2 (IL-2)-stimulated Nb2 cell proliferation is coupled to the activation of p21ras and mitogen-activated protein (MAP) kinase. Stimulation of Nb2-11 cells, growth-arrested in the early G1 phase of the cell cycle, with PRL or IL-2 rapidly (5-10 min) provoked GTP binding to Ras, enhanced tyrosyl phosphorylation of MAP kinase, significantly increased its enzymatic activity, and caused its nuclear translocation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase C, similarly activated Ras and MAP kinase but failed to cause its nuclear translocation. Tyrosine kinase antagonism with genistein inhibited PRL-stimulated Ras and MAP kinase activation. In other experiments, Ras and MAP kinase were each found to be constitutively active in the Nb2-SFJCD1 line. The addition of PRL to these cultures enhanced the activity of these signaling proteins. Finally, the effects of PRL, IL-2, TPA, and phosphatase inhibition on Nb2-11 cell population density and [3H]thymidine uptake were compared. The addition of PRL, IL-2, and TPA significantly stimulated[3H] thymidine incorporation, while only the polypeptide growth factors augmented cell density. Phosphatase inhibition had no effect on either parameter. These results indicate that Nb2 cell proliferation is associated with the early activation of Ras and MAP kinase. Moreover, tyrosyl phosphorylation upstream of Ras activation appears to be required for its subsequent stimulation of mediators, which activate MAP kinase. Protein kinase C activation may be coupled to MAP kinase activation but is not sufficient for Nb2 cell proliferation. PMID:8845300

  10. Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells.

    TOXLINE Toxicology Bibliographic Information

    Rao YP; Buckley DJ; Buckley AR

    1995-10-01

    Several serine/threonine and tyrosine kinase signal transduction pathways have been recently linked to prolactin (PRL) action in lymphoid cells. Utilizing the lactogen-dependent, rat pre-T lymphoma cell line, Nb2-11, and the autonomous subline, Nb2-SFJCD1, studies were conducted to determine whether PRL- or interleukin-2 (IL-2)-stimulated Nb2 cell proliferation is coupled to the activation of p21ras and mitogen-activated protein (MAP) kinase. Stimulation of Nb2-11 cells, growth-arrested in the early G1 phase of the cell cycle, with PRL or IL-2 rapidly (5-10 min) provoked GTP binding to Ras, enhanced tyrosyl phosphorylation of MAP kinase, significantly increased its enzymatic activity, and caused its nuclear translocation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase C, similarly activated Ras and MAP kinase but failed to cause its nuclear translocation. Tyrosine kinase antagonism with genistein inhibited PRL-stimulated Ras and MAP kinase activation. In other experiments, Ras and MAP kinase were each found to be constitutively active in the Nb2-SFJCD1 line. The addition of PRL to these cultures enhanced the activity of these signaling proteins. Finally, the effects of PRL, IL-2, TPA, and phosphatase inhibition on Nb2-11 cell population density and [3H]thymidine uptake were compared. The addition of PRL, IL-2, and TPA significantly stimulated[3H] thymidine incorporation, while only the polypeptide growth factors augmented cell density. Phosphatase inhibition had no effect on either parameter. These results indicate that Nb2 cell proliferation is associated with the early activation of Ras and MAP kinase. Moreover, tyrosyl phosphorylation upstream of Ras activation appears to be required for its subsequent stimulation of mediators, which activate MAP kinase. Protein kinase C activation may be coupled to MAP kinase activation but is not sufficient for Nb2 cell proliferation.

  11. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3 mutation-positive leukemia

    PubMed Central

    Agarwal, Anupriya; MacKenzie, Ryan J.; Eide, Christopher A.; Davare, Monika A.; Watanabe-Smith, Kevin; Tognon, Cristina E.; Mongoue-Tchokote, Solange; Park, Byung; Braziel, Rita M.; Tyner, Jeffrey W.; Druker, Brian J.

    2014-01-01

    To understand the role for cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis we designed a functional RNAi screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen we identified interleukin-2 gamma receptor (IL2R?) as a critical growth determinant for the JAK3A572V mutation-positive AML cell line. We observed that knockdown of IL2R? abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2R? in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2R? completely abrogated the clonogenic potential of JAK3A572V as well as the transforming potential of additional JAK3 activating mutations such as JAK3M511I. In addition, mutation at the IL2R? interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2R? contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant but not wild type JAK3 increased the expression of IL2R?, indicating IL2R? and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall we demonstrate a novel role for IL2R? in potentiating oncogenesis in the setting of JAK3-mutation positive leukemia. Additionally, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth. PMID:25109334

  12. Evidence for a Structural Motif in Toxins and Interleukin-2 That May Be Responsible for Binding to Endothelial Cells and Initiating Vascular Leak Syndrome

    NASA Astrophysics Data System (ADS)

    Baluna, Roxana; Rizo, Josep; Gordon, Brian E.; Ghetie, Victor; Vitetta, Ellen S.

    1999-03-01

    The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

  13. Immunofluorescence techniques for the identification of immune effector cells in rat heart: applications to the study of the myocarditis induced by interleukin-2.

    PubMed

    Yamamoto, A; Wenthold, R J; Zhang, J; Herman, E H; Ferrans, V J

    1995-01-01

    A detailed description is presented of immunohistochemical methods for identification of various types of immune effector cells in rat heart, involving the use of antibodies conjugated with different fluorochromes for the simultaneous demonstration of 2 or 3 different antigens by means of fluorescence microscopy. The initial results of the application of these techniques to the study of the myocarditis induced by interleukin-2 (IL-2) are also presented. Antibodies used included: OX6 antibody (for MHC class II molecules, mainly expressed by dendritic cells): W3/25 and OX8 antibody, for the demonstration of the rat equivalents of CD5 and CD8, respectively: asialo-GM1 ganglioside antibody for the identification of natural killer (NK) cells and lymphokine activated killer (LAK) cells, and ED2 antibody for labeling of macrophages. Fluorochromes used were: fluorescein isothiocyanate (green), tetramethylrhodamine isothiocyanate (red), Texas red sulfonyl chloride (red), and 7-amino-4-methylcoumarin-3-acetic acid (blue). IL-2-induced myocarditis was characterized histologically by infiltration of the myocardium by mononuclear inflammatory cells, microvascular alteration, interstitial edema, and myocyte damage and necrosis. In the initial stages, NK/LAK cells were the predominant type of infiltrating lymphocytes; however, the numbers of these cells decreased sharply in subsequent stages. Macrophages also were initially abundant, and continued to be prevalent throughout the late stages. CD8+ lymphocytes were more numerous than CD4+ lymphocytes. Dendritic-cells showed a diffuse increase in number and also accumulated around foci of myocyte necrosis. Three phenotypes of dendritic cells were recognized, and the possible implications of these findings are discussed. It is hoped that these techniques will prove useful for the immunohistochemical evaluation of various inflammatory diseases of the heart. PMID:7539083

  14. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  15. N-Farnesyloxy-norcantharimide inhibits progression of human leukemic Jurkat T cells through regulation of mitogen-activated protein kinase and interleukin-2 production

    PubMed Central

    Chang, Ming-Che; Wu, Jin-Yi; Liao, Hui-Fen; Chen, Yu-Jen

    2015-01-01

    This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4 μmol/l, which is 11.14-fold (=15.6÷1.4) smaller than the 15.6 μmol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9 μmol/l, which is 8.17-fold (=1698.0÷207.8) smaller than the 1698.0 μmol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.14÷8.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-α. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future. PMID:26288134

  16. N-Farnesyloxy-norcantharimide inhibits progression of human leukemic Jurkat T cells through regulation of mitogen-activated protein kinase and interleukin-2 production.

    PubMed

    Chang, Ming-Che; Wu, Jin-Yi; Liao, Hui-Fen; Chen, Yu-Jen; Kuo, Cheng-Deng

    2015-11-01

    This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4??mol/l, which is 11.14-fold (=15.61.4) smaller than the 15.6??mol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9??mol/l, which is 8.17-fold (=1698.0207.8) smaller than the 1698.0??mol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.148.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-?. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future. PMID:26288134

  17. NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis, ameliorates interleukin 2-induced capillary leakage and reduces tumour growth in adenocarcinoma-bearing mice.

    PubMed Central

    Orucevic, A.; Lala, P. K.

    1996-01-01

    We tested whether NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, can prevent interleukin 2 (IL-2)-induced capillary leakage in tumour-bearing mice without compromising the therapeutic benefits of IL-2. C3H/HeJ female mice transplanted s.c. with 2.5 x 10(5) C3-L5 mammary carcinoma cells were treated with: nothing, IL-2 (ten injections of 15,000 Cetus units i.p. every 8 h), L-NAME (0.1, 0.5, or 1 mg ml-1 drinking water), IL-2 + L-NAME (0.1 or 0.5 or 1 mg ml-1 drinking water). Therapies were given in one round (IL-2, days 10-13; L-NAME, days 9-13) or in two rounds (IL-2, days 10-13 and 20-23; L-NAME, days 9-13 and days 19-23) after tumour transplantation. Capillary leakage was measured from the water contents of the pleural cavities, lungs, spleen and kidneys. Effects of the therapies on the primary tumour size and the number of spontaneous lung metastases were also recorded. NO production was measured as the nitrite + nitrate levels in the serum and in the pleural effusion. After the first round of therapies, addition of L-NAME significantly reduced IL-2-induced pulmonary oedema and water retention in the spleen in a dose-dependent manner. It also significantly reduced the IL-2-induced rise in NO levels in the serum and pleural fluid, but did not affect IL-2-induced pleural effusion or water retention in the kidney. At later stages of tumour growth (day 23), tumours themselves induced significant fluid retention in the lungs and the kidney, which was not aggravated further with the second round of IL-2 therapy. At this time, L-NAME therapy alone ameliorated tumour-induced pulmonary oedema. During both rounds of therapy different doses of L-NAME alone caused a reduction of primary tumour growth as well as spontaneous lung metastases, which improved further with the addition of IL-2. The combination therapy was at least as effective as IL-2 therapy. In summary, L-NAME had anti-tumour effects in vivo, reduced the severity of IL-2-induced capillary leakage in some organs and did not compromise anti-tumour efficacy of IL-2 therapy. Thus, L-NAME could be a valuable adjunct to IL-2-based cancer therapy. PMID:8546905

  18. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor ?-Chain (CD25) Expression Predicts a Poor Prognosis

    PubMed Central

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor ?-chain (IL-2R?, also known as CD25), IL-2R?, IL-3R?, IL-4R?, IL-5R?, IL-6R?, IL-7R?, the common ?-chain (?c), ?c, granulocyte-macrophage colony-stimulating factor (GM-CSF)R?, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3R?, GM-CSFR?, IL-2R?, ?c, c-kit, and G-CSFR exhibited a wide spectrum of ?10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFR? and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2R? in non-M3 patients. Elevated levels of IL-3R?, GM-CSFR?, and IL-2R? correlated with leukocytosis. In patients ?60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2R? was associated with a shorter overall survival. By incorporating IL-2R? status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2R? as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2R? alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ?60 years old. PMID:26375984

  19. Pretreatment with cyclosporine and anti-interleukin 2 receptor antibody abrogates the anti-idiotype response in rat recipients of cardiac allografts.

    PubMed Central

    Tanaka, K; Tilney, N L; Stunkel, K G; Hancock, W W; Diamantstein, T; Kupiec-Weglinski, J W

    1990-01-01

    A 10-day course with ART-18, a mouse monoclonal antibody (mAb) directed against the rat interleukin 2 receptor (IL-2R), prolongs the survival of (LEW x BN)F1 cardiac allografts in LEW recipients to approximately 3 weeks (acute rejection = 8 days, P less than 0.001). We examined host responses against ART-18 idiotype (Id) and mouse immunoglobulin in recipients immunomodulated with ART-18 mAb. Treatment with ART-18 elicited high titers of anti-Id antibodies 14 days after transplantation. However, naive rats given ART-18 before transplantation showed strong anti-Id responses as early as day 4 after engraftment, coinciding with abrogation of the treatment effect (graft survival, approximately 10 days). Preimmunization with irrelevant mouse IgG, which elicited high titers of anti-IgG, did not influence the efficacy of ART-18 upon graft survival (17 days). The use of cyclosporin A (CsA) in conjunction with ART-18 prior to transplantation suppressed the anti-Id response and led to dramatic graft prolongation (greater than 58 days), with two of five grafts surviving indefinitely. This striking effect of CsA plus ART-18 pretreatment did not depend upon CsA per se, as grafts were rejected within 12 days in animals pretreated with CsA alone; in both groups CsA trough levels were comparable. Moreover, administration of CsA before transplantation in concert with control IgG (instead of ART-18) prompted rejection within 2-4 weeks. Thus, discrete interaction(s) between anti-IL-2R mAb and CsA prior to engraftment induces partial host unresponsiveness/tolerance to anti-IL-2R mAb treatment following transplantation and suppresses the neutralizing anti-Id responses, which results in long-term/permanent graft acceptance. This study provides a strategy to overcome the anti-Id response mounted by graft recipients that otherwise limits the efficacy of anti-IL-2R mAb treatment. PMID:2217171

  20. Impairment of the cellular immune response in acute murine toxoplasmosis: regulation of interleukin 2 production and macrophage-mediated inhibitory effects.

    PubMed Central

    Haque, S; Khan, I; Haque, A; Kasper, L

    1994-01-01

    Depression of the cellular immune response to Toxoplasma gondii has been reported in both mice and humans. The present study was undertaken to determine the kinetics and mechanism of the observed downregulation of interleukin 2 (IL-2) production during experimental murine toxoplasmosis. For these investigations, the cell-mediated immune response to the wild type (PTg) was compared with that to the less-virulent mutant parasite (PTgB), which is deficient in the major surface antigen, p30 (SAG-1). Spleen cells from infected A/J mice failed to proliferate in response to Toxoplasma antigens during the first week of infection. Both PTg- and PTgB-infected A/J mice exhibited a significant reduction in the concanavalin A (Con A)-induced lymphoproliferative response. Further, the response of splenocytes from mice infected with the wild-type parasite was significantly diminished compared with that of mice infected with PTgB. The lymphoproliferative response to Con A reached its nadir at day 7 and remained below control levels for at least 14 days postinfection. By day 21 postinfection, the response to Con A and to Toxoplasma antigens was restored to the level observed prior to day 7. Con A-stimulated culture supernatants of spleen cells from mice on day 7 postinfection contained significantly less IL-2 than normal mice. There was no significant difference in the numbers of binding sites or capacity of high-affinity IL-2 receptors between infected and normal mouse splenocytes as determined by Scatchard analysis. Exogenous IL-2 at different concentrations failed to restore the proliferative response of lymphocytes from infected mice to Con A. Adherent macrophages from 7-day-infected mice were able to suppress IL-2 production by normal splenocytes following stimulation with Con A. The inhibitory activity mediated by infected cells was reversed by the antibody to IL-10 but not transforming growth factor beta. There were insignificant levels of nitric oxide production in both infected and normal splenocytes. These results indicate that during acute murine toxoplasmosis, there is a well-defined period (day 7) during which both the T-cell mitogen and parasite antigen-associated lymphoproliferative response are reduced. Further, there is a reduction in the production of IL-2 and an increase in IL-10, which appear to mediate, in part, the observed downregulation of immunity to T. gondii. PMID:8005679

  1. Interleukin-13 is a potent activator of JAK3 and STAT6 in cells expressing interleukin-2 receptor-gamma and interleukin-4 receptor-alpha.

    PubMed Central

    Malabarba, M G; Rui, H; Deutsch, H H; Chung, J; Kalthoff, F S; Farrar, W L; Kirken, R A

    1996-01-01

    The lymphocyte growth factors interleukin-2 (IL2), IL4, IL7, IL9 and IL15 use the common IL2 receptor-gamma (IL2R gamma) and activate the IL2R gamma-associated tyrosine kinase JAK3 (Janus kinase 3). IL13 is structurally related to IL4, competes with IL4 for binding to cell surface receptors and exhibits many similar biological effects. The molecular basis for this functional overlap between IL4 and IL13 has been attributed mainly to a shared use of the 140 kDa IL4R alpha, since these cytokines appear to be uniquely different in that, according to several recent reports, IL13 does not recruit the IL2R gamma or JAK3. This notion has been supported by the identification of a novel 70 kDa IL13 receptor in certain IL13-responsive cell lines that lack IL2R gamma. The present study sheds new light on the issue of functional overlap between IL13 and IL4, by demonstrating for the first time that, in cells that express both IL2R gamma and IL4R alpha, IL13 can mimic IL4-induced heterodimerization of IL2R gamma and IL4R alpha, with consequent marked activation of JAK3 and the transcription factor STAT6 (IL4-STAT). Reconstitution experiments in BA/F3 cells showed that both cytokines require the simultaneous presence of IL4R alpha and IL2R gamma to mediate JAK3 and proliferative responses, and analysis of 12 IL4R alpha variants showed that IL4 and IL13 signals were equally affected by mutations of the cytoplasmic domain. We conclude that IL13 activates the IL2R gamma-associated JAK3 tyrosine kinase in appropriate cell types, and propose that IL13 is capable of interacting with multiple receptor subunits in a cell-dependent and combinatorial manner. Consequently, we predict that partial disruption of IL13 signal transduction also contributes to the severe combined immuno-deficiency syndromes associated with inactivation of the IL2R gamma or JAK3 genes. PMID:8920992

  2. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor ?-Chain (CD25) Expression Predicts a Poor Prognosis.

    PubMed

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor ?-chain (IL-2R?, also known as CD25), IL-2R?, IL-3R?, IL-4R?, IL-5R?, IL-6R?, IL-7R?, the common ?-chain (?c), ?c, granulocyte-macrophage colony-stimulating factor (GM-CSF)R?, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3R?, GM-CSFR?, IL-2R?, ?c, c-kit, and G-CSFR exhibited a wide spectrum of ?10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFR? and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2R? in non-M3 patients. Elevated levels of IL-3R?, GM-CSFR?, and IL-2R? correlated with leukocytosis. In patients ?60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2R? was associated with a shorter overall survival. By incorporating IL-2R? status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2R? as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2R? alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ?60 years old. PMID:26375984

  3. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis

    PubMed Central

    Huntwork-Rodriguez, Sarah; Wang, Bei; Watkins, Trent; Ghosh, Arundhati Sengupta; Pozniak, Christine D.; Bustos, Daisy; Newton, Kim; Kirkpatrick, Donald S.

    2013-01-01

    Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitinproteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults. PMID:23979718

  4. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

    PubMed Central

    Ramphul, Urvashi N.; Garver, Lindsey S.; Molina-Cruz, Alvaro; Canepa, Gaspar E.; Barillas-Mury, Carolina

    2015-01-01

    The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. PMID:25552553

  5. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  6. Biomaterials for mRNA delivery.

    PubMed

    Islam, Mohammad Ariful; Reesor, Emma K G; Xu, Yingjie; Zope, Harshal R; Zetter, Bruce R; Shi, Jinjun

    2015-12-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  7. Analyzing mRNA Expression Using Single mRNA Resolution Fluorescent In Situ Hybridization

    PubMed Central

    Zenklusen, Daniel; Singer, Robert H.

    2011-01-01

    As the product of transcription and the blueprint for translation, mRNA is the main intermediate product of the gene expression pathway. The ability to accurately determine mRNA levels is, therefore, a major requirement when studying gene expression. mRNA is also a target of different regulatory steps, occurring in different subcellular compartments. To understand the different steps of gene expression regulation, it is therefore essential to analyze mRNA in the context of a single cell, maintaining spatial information. Here, we describe a stepwise protocol for fluorescent in situ hybridization (FISH) that allows detection of individual mRNAs in single yeast cells. This method allows quantitative analysis of mRNA expression in single cells, permitting absolute quantification by simply counting mRNAs. It further allows us to study many aspects of mRNA metabolism, from transcription to processing, localization, and mRNA degradation. PMID:20946829

  8. Extraction of mRNA from soil.

    PubMed

    Mettel, Carsten; Kim, Yongkyu; Shrestha, Pravin Malla; Liesack, Werner

    2010-09-01

    Here, we report an efficient method for extracting high-quality mRNA from soil. Key steps in the isolation of total RNA were low-pH extraction (pH 5.0) and Q-Sepharose chromatography. The removal efficiency of humic acids was 94 to 98% for all soils tested. To enrich mRNA, subtractive hybridization of rRNA was most efficient. Subtractive hybridization may be followed by exonuclease treatment if the focus is on the analysis of unprocessed mRNA. The total extraction method can be completed within 8 h, resulting in enriched mRNA ranging from 200 bp to 4 kb in size. PMID:20622132

  9. Translation drives mRNA quality control

    PubMed Central

    Shoemaker, Christopher J.; Green, Rachel

    2015-01-01

    There are three predominant forms of co-translational mRNA surveillance: nonsense-mediated decay (NMD), no-go decay (NGD) and non-stop decay (NSD). While discussion of these pathways often focuses on mRNA fate, there is growing consensus that there are other important outcomes of these processes that must be simultaneously considered. Here, we seek to highlight similarities between NMD, NGD and NSD and their likely origins on the ribosome during translation. PMID:22664987

  10. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  11. Two unique mutations in the interleukin-2 receptor gamma chain gene (IL2RG) cause X-linked severe combined immunodeficiency arising in opposite parental germ lines

    SciTech Connect

    Puck, J.M.; Pepper, A.E.

    1994-09-01

    The gene encoding the gamma chain of the lymphocyte receptor for IL-2 lies in human X13.1 and is mutated in males with X-linked severe combined immunodeficiency (SCID). 27 X-linked SCID mutations have been found in our laboratory. Single strand conformation polymorphism (SSCP) analysis of genomic DNA using primers flanking each of the 8 exons was followed by direct sequencing of abnormally migrating fragments from SCID patients and family members. A 9 bp in-frame duplication insertion was found in IL2RG exon 5 of a patient from a large X-linked SCID pedigree; the resulting duplication of 3 extracellular amino acids, including the first tryptophan of the {open_quotes}WSXWS{close_quotes} cytokine binding motif, is predicted to disrupt interaction of the cytokine receptor chain with its ligand. Genetic linkage studies demonstrated that the grandmaternal X chromosome associated with SCID was contributed to 3 daughters, 2 obligate carriers and 1 woman of unknown status. However, this grandmother`s genomic DNA did not contain the insertion mutation, nor did she have skewed X-chromosome inactivation in her lymphocytes. That both obligate carrier daughters, but not the third daughter, had the insertion proved the grandmother to be a germline mosaic. A second proband had X-linked SCID with a branch point mutation due to substitution of T for A 15 bp 5{prime} of the start of IL2RG exon 3. This mutation resulted in undetectable IL2RG mRNA by Northern blot. Linkage analysis and sequencing of IL2RG DNA in this family proved the mutation to have originated in the germline of the proband`s grandfather, an immunocompetent individual who contributed an X chromosome with normal IL2RG to one daughter and a mutated X to the another.

  12. Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide

    SciTech Connect

    Kaplan, Barbara L.F.; Ouyang Yanli; Herring, Amy; Yea, Sung Su; Razdan, Raj; Kaminski, Norbert E. . E-mail: kamins11@msu.edu

    2005-06-01

    Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-{kappa}B DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.

  13. A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6.

    PubMed Central

    Littaua, R A; Oldstone, M B; Takeda, A; Ennis, F A

    1992-01-01

    A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone. PMID:1370094

  14. Coupling mRNA Synthesis and Decay

    PubMed Central

    Braun, Katherine A.

    2014-01-01

    What has been will be again, what has been done will be done again; there is nothing new under the sun.Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleuscapping, splicing, and polyadenylationare mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasmmRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process. PMID:25154419

  15. Coupling mRNA synthesis and decay.

    PubMed

    Braun, Katherine A; Young, Elton T

    2014-11-15

    What has been will be again, what has been done will be done again; there is nothing new under the sun. -Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus-capping, splicing, and polyadenylation-are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm-mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process. PMID:25154419

  16. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. PMID:25620012

  17. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5? end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  18. Sensitivity of mRNA Translation.

    PubMed

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5' end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  19. mRNA stability in the nucleus*

    PubMed Central

    Liu, Han; Luo, Min; Wen, Ji-kai

    2014-01-01

    Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases (RNases). The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase II in eukaryotes. PMID:24793762

  20. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  1. Control of cytoplasmic mRNA localization.

    PubMed

    Shahbabian, Karen; Chartrand, Pascal

    2012-02-01

    mRNA localization is a mechanism used by various organisms to control the spatial and temporal production of proteins. This process is a highly regulated event that requires multiple cis- and trans-acting elements that mediate the accurate localization of target mRNAs. The intrinsic nature of localization elements, together with their interaction with different RNA-binding proteins, establishes control mechanisms that can oversee the transcript from its birth in the nucleus to its specific final destination. In this review, we aim to summarize the different mechanisms of mRNA localization, with a particular focus on the various control mechanisms that affect the localization of mRNAs in the cytoplasm. PMID:21984598

  2. Gene regulation by mRNA editing

    SciTech Connect

    Ashkenas, J.

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  3. Structure based 3D-QSAR studies of Interleukin-2 inhibitors: Comparing the quality and predictivity of 3D-QSAR models obtained from different alignment methods and charge calculations.

    PubMed

    Halim, Sobia Ahsan; Zaheer-ul-Haq

    2015-08-01

    Interleukin-2 is an essential cytokine in an innate immune response, and is a promising drug target for several immunological disorders. In the present study, structure-based 3D-QSAR modeling was carried out via Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA) methods. Six different partial charge calculation methods were used in combination with two different alignment methods to scrutinize their effects on the predictive power of 3D-QSAR models. The best CoMFA and CoMSIA models were obtained with the AM1 charges when used with co-conformer based substructure alignment (CCBSA) method. The obtained models posses excellent correlation coefficient value and also exhibited good predictive power (for CoMFA: q(2)=0.619; r(2)=0.890; r(2)Pred=0.765 and for CoMSIA: q(2)=0.607; r(2)=0.884; r(2)Pred=0.655). The developed models were further validated by using a set of another sixteen compounds as external test set 2 and both models showed strong predictive power with r(2)Pred=>0.8. The contour maps obtained from these models better interpret the structure activity relationship; hence the developed models would help to design and optimize more potent IL-2 inhibitors. The results might have implications for rational design of specific anti-inflammatory compounds with improved affinity and selectivity. PMID:26051521

  4. Interleukin 2 Induces CD8^+ T Cell-Mediated Suppression of Human Immunodeficiency Virus Replication in CD4^+ T Cells and This Effect Overrides Its Ability to Stimulate Virus Expression

    NASA Astrophysics Data System (ADS)

    Kinter, Audrey L.; Bende, Steven M.; Hardy, Elena C.; Jackson, Robert; Fauci, Anthony S.

    1995-11-01

    The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4^+ T cells by CD8^+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8^+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8^+ T cells. However, IL-2 induces CD8^+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability of IL-2 to induce HIV expression. Five to 25 times fewer CD8^+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25^+) CD8^+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8^+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

  5. The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes.

    PubMed

    Grimm, E A; Wilson, D J

    1985-09-01

    Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies. PMID:3928175

  6. Interleukin-2 receptor antagonist immunosuppression and consecutive viral management in living-donor liver transplantation for human immunodeficiency virus/hepatitis C-co-infected patients: a report of 2 cases.

    PubMed

    Maki, Harufumi; Kaneko, Junichi; Akamatsu, Nobuhisa; Arita, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Tanaka, Tomohiro; Tamura, Sumihito; Sugawara, Yasuhiko; Tsukada, Kunihisa; Kokudo, Norihiro

    2016-02-01

    Management of immunosuppression for human immunodeficiency virus/hepatitis C (HIV/HCV) in living-donor liver transplantation (LDLT) has not been established. We performed LDLT for two patients with HIV/HCV-co-infected end-stage liver disease. The immunosuppression protocol consisted of early calcineurin inhibitor-free and interleukin-2 receptor antagonist (IL2Ra) induction and methylprednisolone. Maintenance low-dose tacrolimus was started and anti-retroviral therapy for HIV was re-started 1week after LDLT. Consecutively, pegylated interferon and ribavirin therapy were successfully added as pre-emptive therapy for HCV. HIV-RNA and HCV-RNA were undetectable on anti-retroviral therapy and HCV treatment at 17 and 8months after LDLT, respectively, with normal liver function. This study is the first report of early calcineurin inhibitor-free and IL2Ra induction with methylprednisolone immunosuppression in LDLT for HIV/HCV-co-infected patients with a favorable outcome. Consecutive HIV/HCV treatment was well tolerated. PMID:26661842

  7. Peripheral blood lymphocytes of patients with common variable immunodeficiency (CVI) produce reduced levels of interleukin-4, interleukin-2 and interferon-gamma, but proliferate normally upon activation by mitogens.

    PubMed Central

    Pastorelli, G; Roncarolo, M G; Touraine, J L; Peronne, G; Tovo, P A; de Vries, J E

    1989-01-01

    Peripheral blood lymphocytes (PBL) of 11 patients with CVI produced reduced levels of interleukin-4 (IL-4) upon activation by mitogens as compared with those secreted by PBL of healthy donors. The interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by PBL of a series of 15 patients with CVI was also reduced. Decreased levels of IL-4 or IL-2 and IFN-gamma production were not only observed after activation by phytohaemagglutinin (PHA) at concentrations of 10 and 1 micrograms/ml, but also after activation by concanavalin A (Con A, 10 micrograms/ml). Longitudinal studies indicated that this defective lymphokine production was consistent upon testing periods up to 5 months. No correlation between reduced IL-4, IL-2 or IFN-gamma production was observed. PBL of patients that produced reduced levels of one lymphokine generally secreted normal levels of the other two lymphokines. Despite the reduced synthesis of the T cell growth factors IL-2 and IL-4, the proliferative responses of the PBL of the patients were in the normal range, which is compatible with the finding that IL-2 and IL-4 have synergistic effects on lymphocyte proliferation, particularly when one of these lymphokines is present at suboptimal concentrations. Since IL-2, IL-4 and IFN-gamma can act as B cell growth and differentiation factors, our data suggest that the reduced synthesis of these lymphokines may contribute to the deficient immunoglobulin production in patients with CVI. PMID:2515013

  8. Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65 (Hsp65) with human interleukin-2 against Mycobacterium tuberculosis in BALB/c mice.

    PubMed

    Wang, Li-Mei; Bai, Yin-Lan; Shi, Chang-Hong; Gao, Hui; Xue, Ying; Jiang, Hong; Xu, Zhi-Kai

    2008-12-01

    Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin-2 (hIL-2) in BALB/c mice. We showed that the DNA vaccine pcDNA-Hsp65-hIL-2 could induce high levels of antigen-specific antibody, IFN-gamma, CD4(+) and CD8(+) T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG. PMID:19133010

  9. [A case of multiple lung metastases and cervical lymph node metastasis of renal cell carcinoma failing to respond to interferon-alpha (IFN-alpha) but markedly responding to interleukin-2 (IL-2)].

    PubMed

    Nishikimi, Toshinori; Ishida, Ryo; Yamada, Hiroshi; Yokoi, Keisuke; Kobayashi, Hiroaki; Obata, Kouji

    2004-01-01

    A 59-year-old man was admitted to our hospital with a left renal mass. A tumor was removed by radical nephrectomy and histological examination revealed renal cell carcinoma (pT2 N0 V1a). Two years later, CT scan showed multiple lung metastases. Despite treatment with recombinant IFN-alpha 2b, 5-FU, and MMC, the disease showed slow progression. About three years after the start of combination therapy, cervical lymph node metastasis appeared. Administration of interleukin-2 (IL-2) was attempted. Intravenous IL-2 therapy was started at a low daily dose of 35 x 10(4) JRU, and the daily dose was increased to 140 x 10(4) JRU. Because of side effect, the dose was subsequently decreased to 70 x 10(4) JRU three times per week. After 31 weeks of IL-2 therapy, his multiple lung metastases and cervical lymph node metastasis disappeared. The patient's natural killer cell (NK) activity and Lymphokine activated killer cell (LAK) activity were low before IL-2 therapy, but both NK activity and LAK activity showed a marked increase after IL-2 therapy started. Therefore, the tumor response to IL-2 was suggested to depend on NK activity and LAK activity. PMID:14978942

  10. Effects of dietary polyunsaturated fatty acids on in vivo splenic cytokine mRNA expression in layer chicks immunized with Salmonella typhimurium lipopolysaccharide.

    PubMed

    Sijben, J W; Schrama, J W; Parmentier, H K; van der Poel, J J; Klasing, K C

    2001-08-01

    Effects of dietary polyunsaturated fatty acids (PUFA) on immune responses in poultry have been reported. However, effects on the underlying mechanisms, such as the role of cytokines, have not been documented because the necessary tools were lacking. Recently, primer sets for chicken interleukin (IL)-1beta, IL-2, interferon-gamma (IFN-gamma), myelomonocytic growth factor (MGF), and transforming growth factor (TGF)-beta2 have become available. Therefore, in the present study we first examined the in vivo effects of an inflammatory challenge with Salmonella typhimurium lipopolysaccharide (LPS) on cytokine profiles in growing laying-type chicks. Second, we examined whether dietary fat sources affected the observed cytokine profiles. Two hundred forty chicks were assigned in a 2 x 4 factorial design of treatments, with injection with LPS or saline and dietary fat source as factors. Factors were i.v. injection with S. typhimurium LPS or saline (control) and four dietary fat sources: corn oil, linseed oil, menhaden oil, and tallow. Two hours after injection, birds were killed, and their spleens were removed for RNA extraction. Reverse transcription polymerase chain reactions with primer sets for chicken IL-1beta, IL-2, IFN-gamma, MGF, TGF-beta2, and beta-actin were performed with RNA samples pooled by pen. The expression of cytokine mRNA was expressed relative to the level of beta-actin mRNA. Interleukin-1 (P < 0.001), MGF (P < 0.0001), IL-2 (P < 0.001), and IFN-gamma (P < 0.001) mRNA expressions were enhanced by challenge with LPS. Immunization treatment had no effect on TGF-beta2 or beta-actin expression. Dietary treatment did not affect mRNA expression of IL-1, MGF, IFN-gamma, TGF-beta2, or beta-actin. Interleukin-2 expression in LPS-injected birds that were fed the fish-oil-enriched diet was enhanced (P = 0.05). The present study indicates that in vivo effects of immune challenge on cytokine mRNA expression can be measured in poultry. The observation that mRNA level of IL-2, but not the mRNA levels of IFN-gamma or MGF, is enhanced by dietary fish oil at 2 h suggests that dietary PUFA at this moment initially affected nave T lymphocytes. PMID:11495469

  11. [Interleukin-2 receptor blockers in renal transplantation].

    PubMed

    Basi?-Juki?, Nikolina; Bubi?-Filipi, Ljubica; Dani?, Ana; Pasini, Josip; Kes, Petar

    2004-01-01

    The last two decades have witnessed significant advances in the renal transplantation immunosuppressive protocols. The introduction of mycophenolate mofetil, tacrolimus, sirolimus and polyclonal antibodies has significantly improved graft survival. However, intensification of immunosuppression results in complications such as malignant diseases, opportunistic infections and metabolic disturbances with consequential increase in cardiovascular mortality. Advances in molecular engineering have made possible the development of monoclonal humanized or chimeric antibodies, which will not induce the host immune response with production of neutralizing antibodies or serum sickness. Antibodies directed against the alpha chain of human IL-2 receptor have recently been introduced into immunosuppressive protocols. Daclizumab is a humanized antibody, and basiliximab is a chimeric antibody, engineered by cloning segments of the murine immunoglobulin sequence into the human-immunoglobulin gene. It decreases immunogenicity while maintaining high specificity for IL-2R alpha chain. The efficacy and safety of both preparations have been reported in large randomized studies. Their use in induction resulted in a significant decrease in acute graft rejections after renal transplantation. The possibility of decreasing the dose or complete withdrawal of certain immunosuppressive agents with the use of IL-2R blockers seems promising for further improvement in the longterm graft survival. Longterm follow-up is necessary to determine their role in solid organ transplantation. PMID:15756806

  12. Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα.

    PubMed

    Dijkstra, Johannes M; Takizawa, Fumio; Fischer, Uwe; Friedrich, Maik; Soto-Lampe, Veronica; Lefèvre, Christophe; Lenk, Matthias; Karger, Axel; Matsui, Taei; Hashimoto, Keiichiro

    2014-02-01

    Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine. PMID:24276591

  13. Anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

    SciTech Connect

    Mule, J.J.; Yang, J.; Shu, S.; Rosenberg, S.A.

    1986-05-15

    Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver. We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus, the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity.

  14. Intranasal immunization against herpes simplex virus infection by using a recombinant glycoprotein D fused with immunomodulating proteins, the B subunit of Escherichia coli heat-labile enterotoxin and interleukin-2.

    PubMed Central

    Hazama, M; Mayumi-Aono, A; Miyazaki, T; Hinuma, S; Fujisawa, Y

    1993-01-01

    To establish a novel strategy of mucosal immunization against herpes simplex virus type 1 (HSV-1) infection, we studied the immune responses elicited by intranasal immunization with several forms of a recombinant glycoprotein D (gD) of HSV-1. A truncated gD (t-gD) co-administered with heat-labile enterotoxin B subunit (LTB) from Escherichia coli induced both a mucosal immune response involving secretion of anti-gD IgA and serum IgG production. The levels of these responses are comparable to those in mice which have recovered from intranasal HSV-1 infections. The fusion protein (t-gD-LTB), consisting of t-gD and LTB, induced the responses more efficiently than did co-administration of t-gD and LTB, although GM1 ganglioside binding activity was significantly reduced in t-gD-LTB. We found that another fusion protein, consisting of t-gD and human interleukin-2 (t-gD-IL-2), also elicited antibody responses comparable to those induced by t-gD-LTB. Immunity acquired by intranasal immunization with t-gD-IL-2 protected mice from intraperitoneal HSV-1 infections, whereas t-gD-LTB or t-gD alone failed to provide protection against infection. Even in a mouse strain that responded highly to subcutaneously administered gD, intranasally administered t-gD did not elicit antibody responses. The lack of response to gD was clearly abrogated by co-administration with IL-2, and administration of t-gD-IL-2 induced an excellent level of antibody responses in this strain. These results suggest that the IL-2 fusion strategy yields a new type of mucosal immunization, the mechanism of which differs from that speculated for the mucosal adjuvant activity of LTB. Images Figure 1 PMID:8388365

  15. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  16. Myc and mRNA capping.

    PubMed

    Dunn, Sianadh; Cowling, Victoria H

    2015-05-01

    c-Myc is upregulated in response to growth factors and transmits the signal to proliferate by altering the gene expression landscape. When genetic alterations result in growth factor-independent c-Myc expression, it can become an oncogene. The majority of human tumour types exhibit a degree of c-Myc deregulation, resulting in unrestrained cell proliferation. c-Myc binds proximal to the promoter region of genes and recruits co-factors including histone acetyltransferases and RNA pol II kinases, which promote transcription. c-Myc also promotes formation of the cap structure at the 5' end of mRNA. The cap is 7-methylguanosine linked to the first transcribed nucleotide of RNA pol II transcripts via a 5' to 5' triphosphate bridge. The cap is added to the first transcribed nucleotide by the capping enzymes, RNGTT and RNMT-RAM. During the early stages of transcription, the capping enzymes are recruited to RNA pol II phosphorylated on Serine-5 of the C-terminal domain. The mRNA cap protects transcripts from degradation during transcription and recruits factors which promote RNA processing including, splicing, export and translation initiation. The proportion of transcripts with a cap structure is increased by elevating c-Myc expression, resulting in increased rates of translation. c-Myc promotes capping by promoting RNA pol II phosphorylation and by upregulating the enzyme SAHH which neutralises the inhibitory bi-product of methylation reactions, SAH. c-Myc-induced capping is required for c-Myc-dependent gene expression and cell proliferation. Targeting capping may represent a new therapeutic opportunity to inhibit c-Myc function in tumours. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. PMID:24681440

  17. Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

    PubMed Central

    Külshammer, Eva; Mundorf, Juliane; Kilinc, Merve; Frommolt, Peter; Wagle, Prerana; Uhlirova, Mirka

    2015-01-01

    ABSTRACT Cancer initiation and maintenance of the transformed cell state depend on altered cellular signaling and aberrant activities of transcription factors (TFs) that drive pathological gene expression in response to cooperating genetic lesions. Deciphering the roles of interacting TFs is therefore central to understanding carcinogenesis and for designing cancer therapies. Here, we use an unbiased genomic approach to define a TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). We show that malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with RasV12 in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, our study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. PMID:26398940

  18. Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.

    PubMed

    Paul, Manoj; Hemshekhar, Mahadevappa; Thushara, Ram M; Sundaram, Mahalingam S; NaveenKumar, Somanathapura K; Naveen, Shivanna; Devaraja, Sannaningaiah; Somyajit, Kumar; West, Robert; Basappa; Nayaka, Siddaiah C; Zakai, Uzma I; Nagaraju, Ganesh; Rangappa, Kanchugarakoppal S; Kemparaju, Kempaiah; Girish, Kesturu S

    2015-01-01

    Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions. PMID:26083398

  19. Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

    PubMed Central

    Paul, Manoj; Hemshekhar, Mahadevappa; Thushara, Ram M.; Sundaram, Mahalingam S.; NaveenKumar, Somanathapura K.; Naveen, Shivanna; Devaraja, Sannaningaiah; Somyajit, Kumar; West, Robert; Basappa; Nayaka, Siddaiah C.; Zakai, Uzma I.; Nagaraju, Ganesh; Rangappa, Kanchugarakoppal S.; Kemparaju, Kempaiah; Girish, Kesturu S.

    2015-01-01

    Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions. PMID:26083398

  20. Cocaine Induces Nuclear Export and Degradation of Neuronal Retinoid X Receptor-? via a TNF-?/JNK-Mediated Mechanism

    PubMed Central

    Kovalevich, Jane; Yen, William; Ozdemir, Ahmet; Langford, Dianne

    2015-01-01

    Cocaine abuse represents an immense societal health and economic burden for which no effective treatment currently exists. Among the numerous intracellular signaling cascades impacted by exposure to cocaine, increased and aberrant production of pro-inflammatory cytokines in the CNS has been observed. Additionally, we have previously reported a decrease in retinoid-X-receptor-gamma (RXR-?) in brains of mice chronically exposed to cocaine. Through obligate heterodimerization with a number of nuclear receptors, RXRs serve as master regulatory transcription factors, which can potentiate or suppress expression of a wide spectrum of genes. Little is known about the regulation of RXR levels, but previous studies indicate cellular stressors such as cytokines negatively regulate levels of RXRs in vitro. To evaluate the mechanism underlying the cocaine-induced decreases in RXR-? levels observed in vivo, we exposed neurons to cocaine in vitro and examined pathways which may contribute to disruption in RXR signaling, including activation of stress pathways by cytokine induction. In these studies, we provide the first evidence that cocaine exposure disrupts neuronal RXR-? signaling in vitro by promoting its nuclear export and degradation. Furthermore, we demonstrate this effect may be mediated, at least in part, by cocaine-induced production of TNF-? and its downstream effector c-Jun-NH-terminal kinase (JNK). Findings from this study are therefore applicable to both cocaine abuse and to pathological conditions characterized by neuroinflammatory factors, such as neurodegenerative disease. PMID:25586717

  1. Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy.

    PubMed

    Klshammer, Eva; Mundorf, Juliane; Kilinc, Merve; Frommolt, Peter; Wagle, Prerana; Uhlirova, Mirka

    2015-10-01

    Cancer initiation and maintenance of the transformed cell state depend on altered cellular signaling and aberrant activities of transcription factors (TFs) that drive pathological gene expression in response to cooperating genetic lesions. Deciphering the roles of interacting TFs is therefore central to understanding carcinogenesis and for designing cancer therapies. Here, we use an unbiased genomic approach to define a TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (Ras(V12)) and loss of the tumor suppressor Scribble (scrib(1)). We show that malignant transformation of the ras(V12)scrib(1) tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to ras(V12)scrib(1) tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in ras(V12)scrib(1) tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like ras(V12)scrib(1) tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While ras(V12)ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, our study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. PMID:26398940

  2. Overexpression of interleukin-2 receptor alpha in a human squamous cell carcinoma of the head and neck cell line is associated with increased proliferation, drug resistance, and transforming ability.

    PubMed

    Kuhn, Deborah J; Smith, David M; Pross, Seth; Whiteside, Theresa L; Dou, Q Ping

    2003-07-01

    It has been previously demonstrated that human carcinomas express interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains. The beta and gamma chains of IL-2R have intermediate binding affinity for IL-2 and are responsible for the intracellular signaling cascades after IL-2 stimulation. IL-2Ralpha lacks the cytoplasmic domain, but is essential for increasing the IL-2-binding affinity of other receptors. Overexpression of IL-2Ralpha in tumor cells is associated with tumor progression and a poor patient prognosis. To define molecular mechanisms responsible for the effects associated with IL-2Ralpha expression, ex vivo experiments were performed with the squamous cell carcinoma head-and-neck cancer line, PCI-13, which was genetically engineered to overexpress the IL-2Ralpha chain. While IL-2Ralpha-overexpressing PCI-13 cells were capable of forming colonies in soft agar, PCI-13 cells transfected with the control vector or those expressing IL-2Rgamma did not. Consistently, IL-2Ralpha-expressing tumor cells proliferated more rapidly than the control or IL-2Rgamma+ cells, associated with increased levels of cyclins A and D1 and cyclin-dependent kinase (cdk(s)) 2 and 4 proteins. In addition, IL-2Ralpha-expressing cells were significantly more resistant to apoptosis induction by a tripeptidyl proteasome inhibitor (ALLN) and two chemotherapeutic drugs (VP-16 and taxol) than the control or IL-2Rgamma+ cells. Accompanying the drug resistance, high levels of anti-apoptotic Bcl-X(L) and Bcl-2 proteins were found in the mitochondria-containing fraction of IL-2Ralpha-expressing tumor cells. Treatment of IL-2Ralpha-expressing cells with a specific Janus kinase 3 (Jak3) inhibitor decreased expression of cyclin A, cyclin D1, Bcl-X(L), and Bcl-2 proteins. Finally, high levels of ubiquitinated proteins were detected in the proliferating IL-2Ralpha-expressing cells. Our data suggest that increased proliferation rates and decreased drug sensitivity of IL-2Ralpha-expressing tumor cells are responsible for the enhanced tumor aggressiveness and poor clinical prognosis of patients whose tumors express IL-2Ralpha. PMID:12858347

  3. Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein.

    PubMed Central

    Gilks, C B; Bear, S E; Grimes, H L; Tsichlis, P N

    1993-01-01

    During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype. Images PMID:8441411

  4. Influence of thyroxine and thyroxine with growth hormone and prolactin on splenocyte subsets and on the expression of interleukin-2 and prolactin receptors on splenocyte subsets of Snell dwarf mice.

    PubMed

    Gala, R R

    1995-11-01

    A number of immune parameters were examined in Snell dwarf mice and compared with normal littermates. The number of splenocytes per gram of body weight were significantly decreased in dwarf animals, and the decrease was distributed throughout the CD4, CD8, B220, and MAC-1 subsets. The percentage of CD4 and CD8 splenocytes was markedly increased, and the percentage of B220 and MAC-1 splenocytes markedly decreased, in dwarf animals. In addition, the percentage of splenocyte T cells constitutively expressing interleukin-2 (IL-2) receptors and prolactin (PRL) receptors was decreased, with the CD4 subset presenting the most dramatic effect. The effects of replacing the hormones deficient in the Snell dwarf mouse (i.e., growth hormone [GH], prolactin [PRL], and thyroxine [T4] on the above immune parameters were also examined. The administration of T4 alone for 10 days corrected the defect in splenocyte cell numbers per grams body weight for both the CD4 and CD8 subsets, but only partially corrected the defect for the B220 and MAC-1 subsets. The addition of rbGH and rbPRL for the last 3 days of T4 injection had little additive effect on the number of CD4 and CD8 cells but increased the number of B220 and MAC-1 subsets to values comparable to those of normal animals on the basis of body weight. The decrease in the percentage of CD4 splenocytes in dwarf animals constitutively expressing IL-2R was partially corrected by T4 injection and completely corrected by the addition of rbGH and rbPRL for the last 3 days. The decrease in CD4 splenocytes constitutively expressing PRLR was partially corrected by T4 injection alone and the addition of rbGH and rPRL resulted in percentages comparable to that of normal animals. The results indicate that Snell dwarf animals are deficient in immune parameters and that the administration of the hormones lacking in this animal can correct the deficiencies. PMID:7568281

  5. Highly efficient expression of interleukin-2 under the control of rabbit β-globin intron II gene enhances protective immune responses of porcine reproductive and respiratory syndrome (PRRS) DNA vaccine in pigs.

    PubMed

    Du, Yijun; Lu, Yu; Wang, Xinglong; Qi, Jing; Liu, Jiyu; Hu, Yue; Li, Feng; Wu, Jiaqiang; Guo, Lihui; Liu, Junzhen; Tao, Haiying; Sun, Wenbo; Chen, Lei; Cong, Xiaoyan; Ren, Sufang; Shi, Jianli; Li, Jun; Wang, Jinbao; Huang, Baohua; Wan, Renzhong

    2014-01-01

    Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections. PMID:24603502

  6. A phase 1b study of humanized KS-interleukin-2 (huKS-IL2) immunocytokine with cyclophosphamide in patients with EpCAM-positive advanced solid tumors

    PubMed Central

    2013-01-01

    Background Humanized KS-interleukin-2 (huKS-IL2), an immunocytokine with specificity for epithelial cell adhesion molecule (EpCAM), has demonstrated favorable tolerability and immunologic activity as a single agent. Methods Phase 1b study in patients with EpCAM-positive advanced solid tumors to determine the maximum tolerated dose (MTD) and safety profile of huKS-IL2 in combination with low-dose cyclophosphamide. Treatment consisted of cyclophosphamide (300 mg/m2 on day 1), and escalating doses of huKS-IL2 (0.5–4.0 mg/m2 IV continuous infusion over 4 hours) on days 2, 3, and 4 of each 21-day cycle. Safety, pharmacokinetic profile, immunogenicity, anti-tumor and biologic activity were evaluated. Results Twenty-seven patients were treated for up to 6 cycles; 26 were evaluable for response. The MTD of huKS-IL2 in combination with 300 mg/m2 cyclophosphamide was 3.0 mg/m2. At higher doses, myelosuppression was dose-limiting. Transient lymphopenia was the most common grade 3/4 adverse event (AE). Other significant AEs included hypotension, hypophosphatemia, and increase in serum creatinine. All patients recovered from these AEs. The huKS-IL2 exposure was dose-dependent, but not dose-proportional, accumulation was negligible, and elimination half-life and systemic clearance were independent of dose and time. Most patients had a transient immune response to huKS-IL2. Immunologic activity was observed at all doses. Ten patients (38%) had stable disease as best response, lasting for ≥ 4 cycles in 3 patients. Conclusion The combination of huKS-IL2 with low-dose cyclophosphamide was well tolerated. Although no objective responses were observed, the combination showed evidence of immunologic activity and 3 patients showed stable disease for ≥ 4 cycles. Trial registration http://NCT00132522 PMID:23320927

  7. Visualization of mRNA Localization in Xenopus Oocytes

    PubMed Central

    Gagnon, James A.; Mowry, Kimberly L.

    2011-01-01

    Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. This technique overcomes a significant hurdle of imaging RNA in the frog oocyte while providing a rapid method of visualizing mRNA localization in high resolution. PMID:21431735

  8. mRNA Localization and Translational Control in Drosophila Oogenesis

    PubMed Central

    Lasko, Paul

    2012-01-01

    Localization of an mRNA species to a particular subcellular region can complement translational control mechanisms to produce a restricted spatial distribution of the protein it encodes. mRNA localization has been studied most in asymmetric cells such as budding yeast, early embryos, and neurons, but the process is likely to be more widespread. This article reviews the current state of knowledge about the mechanisms of mRNA localization and its functions in early embryonic development, focusing on Drosophila where the relevant knowledge is most advanced. Links between mRNA localization and translational control mechanisms also are examined. PMID:22865893

  9. mRNA localization in the Drosophila germline

    PubMed Central

    Weil, Timothy T

    2014-01-01

    Localization and the associated translational control of mRNA is a well established mechanism for segregating cellular protein expression. Drosophila has been instrumental in deciphering the prevailing mechanisms of mRNA localization and regulation. This review will discuss the diverse roles of mRNA localization in the Drosophila germline, the cis-elements and cellular components regulating localization and the superimposition of translational regulatory mechanisms. Despite a history of discovery, there are still many fundamental questions regarding mRNA localization that remain unanswered. Take home messages, outstanding questions and future approaches that will likely lead to resolving these unknowns in the future are summarized at the end. PMID:25482896

  10. Functional Integration of mRNA Translational Control Programs.

    PubMed

    MacNicol, Melanie C; Cragle, Chad E; Arumugam, Karthik; Fosso, Bruno; Pesole, Graziano; MacNicol, Angus M

    2015-01-01

    Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease. PMID:26197342

  11. Functional Integration of mRNA Translational Control Programs

    PubMed Central

    MacNicol, Melanie C.; Cragle, Chad E.; Arumugam, Karthik; Fosso, Bruno; Pesole, Graziano; MacNicol, Angus M.

    2015-01-01

    Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease. PMID:26197342

  12. A numbers game underpins cytoplasmic mRNA transport.

    PubMed

    Doyle, Michael; Kiebler, Michael A

    2012-04-01

    Microtubule-based mRNA transport participates in the establishment of cell asymmetries. An in vitro reconstitution assay demonstrates that localization signals present in an mRNA influence motor copy number on single RNA molecule cargoes, ultimately leading to highly polarized distributions of transcripts. PMID:22469827

  13. Surviving hypoxia by modulation of mRNA translation rate

    PubMed Central

    Fähling, Michael

    2009-01-01

    Cells can survive hypoxia/anoxia by metabolic rate depression, which involves lowering of mRNA translation rates in an ATP-dependent manner. By activating anaerobic ATP production (glycolysis), the inhibitory influence on mRNA translation in hypoxia can be abolished. In severe hypoxia, glycolysis cannot fully restore the ATP demand, thus causing a long-lasting inhibition of global protein synthesis. During moderate hypoxia, fermentative ATP production may maintain normal ATP levels. However, an activation of hypoxia tolerance mechanisms, including specific mRNA translation, also takes place. The latter may be attributed to oxygen-dependent (but not ATP dependent) processes such as the activation of the hypoxia-inducible factor cascade. In summary, hypoxia-induced decline in cellular ATP level can be counteracted by suppression of global mRNA translation rate. Sustained protein synthesis seems to be attributed to the activation of specific mRNA translation under long-term hypoxic conditions. PMID:19674191

  14. Changes in Chloroplast mRNA Stability during Leaf Development.

    PubMed Central

    Klaff, P; Gruissem, W

    1991-01-01

    During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D. Decay of psbA and rbcL mRNAs was assayed in young and mature leaves. The psbA mRNA half-life was increased more than twofold in mature leaves compared with young leaves, whereas rbcL mRNA decayed with a similar relative half-life at both leaf developmental stages. The direct in vivo measurements demonstrated that differential mRNA stability in higher plant plastids can account for differences in mRNA accumulation during leaf development. The role of polysome association in mRNA decay was also investigated. Using organelle-specific translation inhibitors that force mRNAs into a polysome-bound state or deplete mRNAs of ribosomes, we measured mRNA decay in vivo in either state. The results showed that rbcL and psbA mRNAs are less stable when bound to polysomes relative to the polysome-depleted mRNAs and that their stabilities are differentially affected by binding to polysomes. The results suggested that ribosome binding and/or translation of the psbA and rbcL mRNAs may function to modulate the rate of their decay in chloroplasts. PMID:12324602

  15. Effects of DNA replication on mRNA noise

    PubMed Central

    Peterson, Joseph R.; Cole, John A.; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A.

    2015-01-01

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript’s half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  16. mRNA quantification via second harmonic super resolution microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Cho, Il-Hoon; Kadam, Ulhas; Irudayaraj, Joseph

    2014-03-01

    Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. However, most available technologies for mRNA detection rely on cell extraction that inherently destroys the tissue context and provide only average expression levels from cell populations or whole tissues. In this paper, we proposed a novel super resolution concept, second harmonic generation (SHG) super-resolution microscopy (SHaSM), and apply that to detect single short mRNA transcript, Her2 mRNA, beyond the diffraction limit. Nano-sized SHG crystals, barium titanium oxide BaTiO3 (BTO), were functionalized with two complimentary strands of Her2 mRNA after the chemical surface-modification. Dimer schematic was used to improve the specificity of detection and quantification, where two BTO monomers bind to the Her2 mRNA to form a dimer and being visualized via the SHaSM. SHaSM is able to detect single BTO nanocrystal with ~20 nm spatial resolution, and differentiate BTO dimers (Her2 mRNA) from BTO monomers (non-specific bounded BTO nanocrystal) with high specificity.

  17. Heritable variation of mRNA decay rates in yeast

    PubMed Central

    Andrie, Jennifer M.; Wakefield, Jon

    2014-01-01

    Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been a subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. We estimate that 31% of genes exhibit allelic differences in mRNA decay rates, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rates have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rates. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay have opposite effects, suggesting that steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels. PMID:25258386

  18. Polyadenylation of Vesicular Stomatitis Virus mRNA

    PubMed Central

    Ehrenfeld, Ellie

    1974-01-01

    Vesicular stomatitis virus (VSV) mRNA isolated from infected cell polysomes contains polyadenylic acid [poly(A)] sequences. Detergent-activated purified virions in vitro can transcribe complementary RNA, which has sedimentation properties similar to mRNA, and this RNA also contains poly(A) sequences. Digestion of virion RNA with U2 RNase under conditions where hydrolysis is specific for purine linkages leaves no sequences of polyuridylic acid corresponding in length to the poly(A) on the transcripts. Growth of infectious virus is not inhibited by 3-deoxyadenosine (cordycepin) under conditions in which it inhibits polyadenylation of cellular mRNA. The virus-specific mRNA produced in the presence of cordycepin has poly(A) sequences of the same size distribution as that synthesized in the absence of cordycepin. PMID:4363251

  19. Signaling Pathways That Control mRNA Turnover

    PubMed Central

    Thapar, Roopa; Denmon, Andria P.

    2013-01-01

    Cells regulate their genomes mainly at the level of transcription and at the level of mRNA decay. While regulation at the level of transcription is clearly important, the regulation of mRNA turnover by signaling networks is essential for a rapid response to external stimuli. Signaling pathways result in posttranslational modification of RNA binding proteins by phosphorylation, ubiquitination, methylation, acetylation etc. These modifications are important for rapid remodeling of dynamic ribonucleoprotein complexes and triggering mRNA decay. Understanding how these posttranslational modifications alter gene expression is therefore a fundamental question in biology. In this review we highlight recent findings on how signaling pathways and cell cycle checkpoints involving phosphorylation, ubiquitination, and arginine methylation affect mRNA turnover. PMID:23602935

  20. Multiple crosstalks between mRNA biogenesis and SUMO.

    PubMed

    Rouvire, Jrme O; Geoffroy, Marie-Claude; Palancade, Benoit

    2013-10-01

    mRNA metabolism involves the orchestration of multiple nuclear events, including transcription, processing (e.g., capping, splicing, polyadenylation), and quality control. This leads to the accurate formation of messenger ribonucleoparticles (mRNPs) that are finally exported to the cytoplasm for translation. The production of defined sets of mRNAs in given environmental or physiological situations relies on multiple regulatory mechanisms that target the mRNA biogenesis machineries. Among other regulations, post-translational modification by the small ubiquitin-like modifier SUMO, whose prominence in several cellular processes has been largely demonstrated, also plays a key role in mRNA biogenesis. Analysis of the multiple available SUMO proteomes and functional validations of an increasing number of sumoylated targets have revealed the key contribution of SUMO-dependent regulation in nuclear mRNA metabolism. While sumoylation of transcriptional activators and repressors is so far best documented, SUMO contribution to other stages of mRNA biogenesis is also emerging. Modification of mRNA metabolism factors by SUMO determine their subnuclear targeting and biological activity, notably by regulating their molecular interactions with nucleic acids or protein partners. In particular, sumoylation of DNA-bound transcriptional regulators interfere with their association to target sequences or chromatin modifiers. In addition, the recent identification of enzymes of the SUMO pathway within specialized mRNA biogenesis machineries may provide a further level of regulation to their specificity. These multiple crosstalks between mRNA metabolism and SUMO appear therefore as important players in cellular regulatory networks. PMID:23584125

  1. Messenger RNA (mRNA) Nanoparticle Tumour Vaccination

    PubMed Central

    Phua, Kyle K.L.; Nair, Smita K.; Leong, Kam W.

    2014-01-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNAs biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research. PMID:24904987

  2. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration

    PubMed Central

    Guan, Shan; Rosenecker, Joseph

    2015-01-01

    During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent. PMID:26352268

  3. Paclitaxel inhibits mRNA transport in axons.

    PubMed

    Bobylev, Ilja; Joshi, Abhijeet R; Barham, Mohammed; Ritter, Christian; Neiss, Wolfram F; Hke, Ahmet; Lehmann, Helmar C

    2015-10-01

    Paclitaxel is an integral component of solid tumor treatment. This chemotherapeutic agent provokes an often irreversible peripheral sensory neuropathy with pathological features of distal axonal degeneration. Current pathological concepts assume that polymerization of axonal microtubules and mitochondrial dysfunction contributes to the development of paclitaxel-induced peripheral neuropathy. The relationship, however, between microtubule stabilization, mitotoxicity and axonal degeneration is still not completely understood. To explore the function of axonal mitochondria we treated transgenic mice that harbor cyan fluorescent protein (CFP)-labeled neuronal mitochondria with repeated doses of paclitaxel and assessed neuropathic changes by nerve conduction and histological studies. In addition, mitochondrial content and morphology was determined by ex vivo imaging of axons containing CFP-labeled mitochondria. Using quantitative RT-PCR and fluorescence-labeled mRNA we determined axonal mRNA transport of nuclear encoded mitochondrial proteins. Prolonged treatment with high doses of paclitaxel-induced a predominant sensory neuropathy in mice. Although mitochondrial velocity in axons per se was not altered, we observed significant changes in mitochondrial morphology, suggesting that paclitaxel treatment impairs the dynamics of axonal mitochondria. These changes were caused by decreased levels of nuclear encoded mRNA, including the mitochondrial fusion/fission machinery. Moreover, impaired axonal mRNA transport in vitro resulted in mitochondrial dysfunction and subsequent axonal degeneration. Taken together, our experiments provide evidence that disrupted axonal transport of nuclear derived mRNA plays a crucial role in the pathogenesis of paclitaxel-induced sensory neuropathy. PMID:26188177

  4. Initiation of mRNA decay in bacteria.

    PubMed

    Laalami, Soumaya; Zig, Lna; Putzer, Harald

    2014-05-01

    The instability of messenger RNA is fundamental to the control of gene expression. In bacteria, mRNA degradation generally follows an "all-or-none" pattern. This implies that if control is to be efficient, it must occur at the initiating (and presumably rate-limiting) step of the degradation process. Studies of E. coli and B. subtilis, species separated by 3 billion years of evolution, have revealed the principal and very disparate enzymes involved in this process in the two organisms. The early view that mRNA decay in these two model organisms is radically different has given way to new models that can be resumed by "different enzymes-similar strategies". The recent characterization of key ribonucleases sheds light on an impressive case of convergent evolution that illustrates that the surprisingly similar functions of these totally unrelated enzymes are of general importance to RNA metabolism in bacteria. We now know that the major mRNA decay pathways initiate with an endonucleolytic cleavage in E. coli and B. subtilis and probably in many of the currently known bacteria for which these organisms are considered representative. We will discuss here the different pathways of eubacterial mRNA decay, describe the major players and summarize the events that can precede and/or favor nucleolytic inactivation of a mRNA, notably the role of the 5' end and translation initiation. Finally, we will discuss the role of subcellular compartmentalization of transcription, translation, and the RNA degradation machinery. PMID:24064983

  5. Protein targeting to subcellular organelles via MRNA localization.

    PubMed

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. PMID:23457718

  6. Nuclear Retention of mRNA in Mammalian Tissues

    PubMed Central

    Bahar Halpern, Keren; Caspi, Inbal; Lemze, Doron; Levy, Maayan; Landen, Shanie; Elinav, Eran; Ulitsky, Igor; Itzkovitz, Shalev

    2015-01-01

    Summary mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. PMID:26711333

  7. Control of Cell Migration Through Mrna Localization and Local Translation

    PubMed Central

    Liao, Guoning; Mingle, Lisa; Van De Water, Livingston; Liu, Gang

    2014-01-01

    Cell migration plays an important role in many normal and pathological functions such as development, wound healing, immune defense and tumor metastasis. Polarized migrating cells exhibit asymmetric distribution of many cytoskeletal proteins which is believed to be critical for establishing and maintaining cell polarity and directional cell migration. To target these proteins to the site of function, cells use a variety of mechanisms such as protein transport and mRNA localization-mediated local protein synthesis. In contrast to the former which is intensively investigated and relatively well understood, the latter has been under-studied and relatively poorly understood. However, recent advances in the study of mRNA localization and local translation have demonstrated that mRNA localization and local translation are specific and effective ways for protein localization and are crucial for embryo development, neuronal function and many other cellular processes. There are excellent reviews on mRNA localization, transport and translation during development and other cellular processes. This review will focus on mRNA localization-mediated local protein biogenesis and its impact on somatic cell migration. PMID:25264217

  8. Nuclear Retention of mRNA in Mammalian Tissues.

    PubMed

    Bahar Halpern, Keren; Caspi, Inbal; Lemze, Doron; Levy, Maayan; Landen, Shanie; Elinav, Eran; Ulitsky, Igor; Itzkovitz, Shalev

    2015-12-29

    mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. PMID:26711333

  9. Endoribonucleases--enzymes gaining spotlight in mRNA metabolism.

    PubMed

    Li, Wai Ming; Barnes, Tavish; Lee, Chow H

    2010-02-01

    The efficient turnover of messenger RNA represents an important mechanism that allows the cell to control gene expression. Until recently, the mechanism of mRNA decay was mainly attributed to exonucleases, comprising enzymes that degrade RNAs from the ends of the molecules. This article summarizes the endoribonucleases, comprising enzymes that cleave RNA molecules internally, which were identified in more recent years in eukaryotic mRNA metabolism. Endoribonucleases have received little attention in the past, based on the difficulty in their identification and a lack of understanding of their physiological significance. This review aims to compare the similarities and differences among this group of enzymes, as well as their known cellular functions. Despite the many differences in protein structure, and thus difficulties in identifying them based on amino acid sequence, most endoribonucleases possess essential cellular functions and have been shown to play an important role in mRNA turnover. PMID:19968858

  10. Effect of ribosome shielding on mRNA stability

    NASA Astrophysics Data System (ADS)

    Deneke, Carlus; Lipowsky, Reinhard; Valleriani, Angelo

    2013-08-01

    Based on the experimental evidence that translating ribosomes stabilize the mRNAs, we introduce and study a theoretical model for the dynamic shielding of mRNA by ribosomes. We present an improved fitting of published decay assay data in E. coli and show that only one third of the decay patterns are exponential. Our new transcriptome-wide estimate of the average lifetimes and mRNA half-lives shows that these timescales are considerably shorter than previous estimates. We also explain why there is a negative correlation between mRNA length and average lifetime when the mRNAs are subdivided in classes sharing the same degradation parameters. As a by-product, our model indicates that co-transcriptional translation in E. coli may be less common than previously believed.

  11. Interplay between viruses and host mRNA degradation

    PubMed Central

    Narayanan, Krishna; Makino, Shinji

    2013-01-01

    Messenger RNA degradation is a fundamental cellular process that plays a critical role in regulating gene expression by controlling both the quality and the abundance of mRNAs in cells. Naturally, viruses must successfully interface with the robust cellular RNA degradation machinery to achieve an optimal balance between viral and cellular gene expression and establish a productive infection in the host. In the past several years, studies have discovered many elegant strategies that viruses have evolved to circumvent the cellular RNA degradation machinery, ranging from disarming the RNA decay pathways and co-opting the factors governing cellular mRNA stability to promoting host mRNA degradation that facilitates selective viral gene expression and alters the dynamics of host-pathogen interaction. This review summarizes the current knowledge of the multifaceted interaction between viruses and cellular mRNA degradation machinery to provide an insight into the regulatory mechanisms that influence gene expression in viral infections. PMID:23274304

  12. Mechanism of mRNA binding to bovine mitochondrial ribosomes.

    PubMed

    Denslow, N D; Michaels, G S; Montoya, J; Attardi, G; O'Brien, T W

    1989-05-15

    The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria. PMID:2542274

  13. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  14. The histone mRNA 3' end is required for localization of histone mRNA to polyribosomes.

    PubMed Central

    Sun, J; Pilch, D R; Marzluff, W F

    1992-01-01

    The final step in mRNA biosynthesis is transport of the mRNA from the nucleus to the cytoplasm. Histone genes from which the 3' stem-loop has been deleted are transcribed to give RNAs with heterogeneous 3' ends. These RNAs are localized in the nucleus and are stable. Addition of the histone 3' processing signal either on short (< 250 nts) or long (> 1000 nts) transcripts restores 3' processing and transport of the mRNA to the cytoplasm. In addition chimeric histone-U1 snRNA genes which produced RNAs with either histone or U1 3' ends were analyzed. Transcripts which ended with U1 snRNA 3' ends were not efficiently localized to polyribosomes. However, transcripts containing the same sequences including the snRNA 3' end followed by the histone 3' end were present in the cytoplasm on polyribosomes. Taken together these results suggest that the histone 3' end is required for export of histone mRNA to the cytoplasm and association of the mRNA with polyribosomes. Images PMID:1461736

  15. Yeast phospholipid biosynthesis is linked to mRNA localization.

    PubMed

    Hermesh, Orit; Genz, Christian; Yofe, Ido; Sinzel, Monika; Rapaport, Doron; Schuldiner, Maya; Jansen, Ralf-Peter

    2014-08-01

    Regulation of the localization of mRNAs and local translation are universal features in eukaryotes and contribute to cellular asymmetry and differentiation. In Saccharomyces cerevisiae, localization of mRNAs that encode membrane proteins requires the She protein machinery, including the RNA-binding protein She2p, as well as movement of the cortical endoplasmic reticulum (cER) to the yeast bud. In a screen for ER-specific proteins necessary for the directional transport of WSC2 and EAR1 mRNAs, we have identified enzymes that are involved in phospholipid metabolism. Loss of the phospholipid methyltransferase Cho2p, which showed the strongest impact on mRNA localization, disturbs mRNA localization, as well as ER morphology and segregation, owing to an increase in the amount of cellular phosphatidylethanolamine (PtdEtn). Mislocalized mRNPs containing She2p colocalize with aggregated cER structures, suggestive of the entrapment of mRNA and She2p by the elevated PtdEtn level. This was confirmed by the elevated binding of She2p to PtdEtn-containing liposomes. These findings underscore the importance of ER membrane integrity in mRNA transport. PMID:24906800

  16. Mapping of the three species of polyoma mRNA.

    PubMed Central

    Trler, H; Salomon, C; Allet, B; Weil, R

    1976-01-01

    The polyoma mRNA's present in the cytoplasm of primary cultures of mouse kidney cells during lytic infection were characterized by sedimentation velocity analysis and by hybridization to polyoma DNA fragments generated by a specific endonuclease of Hemophilus parainfluenzae (Hpa II). Images PMID:179087

  17. Phylogenetic comparison of oskar mRNA localization signals.

    PubMed

    Kim, Jihyun; Lee, Jiyeon; Lee, Sujung; Lee, Borim; Kim-Ha, Jeongsil

    2014-01-31

    As a way to spatially control the expression of genes within cells, RNA localization is being recognized as an important process by which proteins are restricted to specific subcellular domains, which occurs in more diverse types of tissue than previously considered. Although many localized RNAs have been identified, information on cis-acting elements of localization is still limited. As transcripts of oskar (osk) are known to localize to the posterior pole of oocytes, we computationally analyzed a conserved sequence among eight Drosophila species and tested its role as a localization element. Dimerization of osk mRNA did not occur when the motif was deleted, but this did not affect assembly of osk mRNA-containing ribonucleoprotein (RNP) complexes. Without the motif, however, large RNP complex particles accumulated in nurse cells, and only a small fraction of these RNP complexes was transported into oocytes and properly localized to the posterior pole. Therefore, this motif may be required for the early transport of osk mRNA into oocytes. Also, as dimerization of osk mRNA does not seem to be a prerequisite for the assembly of RNP complexes, a dimerization-independent mechanism may also serve to localize osk mRNA to the posterior pole. PMID:24440702

  18. BIOMARKERS OF ENDOCRINE DISRUPTION AT THE MRNA LEVEL

    EPA Science Inventory

    Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for ...

  19. Molecular cloning of human ornithine aminotransferase mRNA

    SciTech Connect

    Inana, G.; Totsuka, S.; Redmond, M.; Dougherty, T.; Nagle, J.; Shiono, T.; Ohura, T. Kominami, E.; Katunuma, N.

    1986-03-01

    The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO/sub 4//PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO/sub 4//PAGE. lambdagt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of /sup 32/P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approx. = 2.2 kilobases.

  20. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    PubMed Central

    Bonnet, Amandine; Palancade, Benoit

    2014-01-01

    Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

  1. Analysis of mRNA recognition by human thymidylate synthase

    PubMed Central

    Brunn, NicholasD.; Dibrov, SergeyM.; Kao, MelodyB.; Ghassemian, Majid; Hermann, Thomas

    2014-01-01

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2?-deoxyuridine-5?-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helixloophelix domain on the protein surface was identified as the putative RNA-binding site. PMID:25423174

  2. Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

    PubMed Central

    Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie A.

    2007-01-01

    Background Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5? and 3? splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. Methodology/Principal Finding Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3? splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. Conclusions/Significance Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exonexon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites. PMID:17487273

  3. Peptide inhibitors of botulinum neurotoxin by mRNA display

    SciTech Connect

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H. . E-mail: yangdc@georgetown.edu

    2005-10-07

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

  4. Measurements of mRNA degradation in Borrelia burgdorferi.

    PubMed

    Archambault, Linda; Borchert, J Simmons; Bergeron, Jennifer; Snow, Santina; Schlax, Paula Jean

    2013-11-01

    The importance of gene regulation in the enzootic cycle of Borrelia burgdorferi, the spirochete that causes Lyme disease, is well established. B. burgdorferi regulates gene expression in response to changes in environmental stimuli associated with changing hosts. In this study, we monitored mRNA decay in B. burgdorferi following transcriptional arrest with actinomycin D. The time-dependent decay of transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), outer surface lipoproteins (ospA and ospC), and a flagellar protein (flaB) have different profiles and indicate half-lives ranging from approximately 1 min to more than 45 min in cells cultured at 35C. Our results provide a first step in characterizing mRNA decay in B. burgdorferi and in investigating its role in gene expression and regulation. PMID:23974029

  5. Developmental expression of choline acetyltransferase mRNA in Drosophila.

    PubMed

    Carbini, L A; Muoz Maines, V J; Salvaterra, P M

    1990-11-01

    We have measured the steady state levels of choline acetyltransferase (ChAT, EC 2.3.1.6) mRNA during different developmental stages of Drosophila melanogaster using a ChAT specific cRNA probe. ChAT mRNA was first detected approximately 6-7 h after oviposition, increased until the 1st-2nd larval instar, decreased into early pupal stages and increased again during late pupation, reaching a maximum in adults. Northern analysis showed a major RNA band with a Mr of 4.7 kilobases and Western analysis also showed a single major 75 kD protein band at all developmental stages. Our results support the hypothesis that a major point of regulation of ChAT expression may be at the transcriptional level. PMID:2128533

  6. The Current Status of Vertebrate Cellular mRNA IRESs

    PubMed Central

    Jackson, Richard J.

    2013-01-01

    Internal ribosome entry sites/segments (IRESs) were first discovered over 20 years ago in picornaviruses, followed by the discovery of two other types of IRES in hepatitis C virus (HCV), and the dicistroviruses, which infect invertebrates. In the meantime, reports of IRESs in eukaryotic cellular mRNAs started to appear, and the list of such putative IRESs continues to grow to the point in which it now stands at ∼100, 80% of them in vertebrate mRNAs. Despite initial skepticism from some quarters, there now seems universal agreement that there is genuine internal ribosome entry on the viral IRESs. However, the same cannot be said for cellular mRNA IRESs, which continue to be shrouded in controversy. The aim of this article is to explain why vertebrate mRNA IRESs remain controversial, and to discuss ways in which these controversies might be resolved. PMID:23378589

  7. Regulation of mRNA Translation by Signaling Pathways

    PubMed Central

    Roux, Philippe P.; Topisirovic, Ivan

    2012-01-01

    mRNA translation is the most energy consuming process in the cell. In addition, it plays a pivotal role in the control of gene expression and is therefore tightly regulated. In response to various extracellular stimuli and intracellular cues, signaling pathways induce quantitative and qualitative changes in mRNA translation by modulating the phosphorylation status and thus the activity of components of the translational machinery. In this work we focus on the phosphoinositide 3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK) pathways, as they are strongly implicated in the regulation of translation in homeostasis, whereas their malfunction has been linked to aberrant translation in human diseases, including cancer. PMID:22888049

  8. Cell biology. TAPping into mRNA export.

    PubMed

    Moore, M J; Rosbash, M

    2001-11-30

    There seem to be numerous pathways for exporting mRNAs from the nucleus to the cytoplasm. But working out which set of export adaptors and receptors transport individual mRNAs has been very difficult. In a Perspective, Moore and Rosbash discuss a new strategy using cell-penetrating peptide inhibitors for unraveling the routes of mRNA export in living cells (Gallouzi and Steitz). PMID:11729289

  9. The utility of protein and mRNA correlation

    SciTech Connect

    Payne, Samuel H.

    2015-01-01

    Transcriptomic, proteomic and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight.

  10. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  11. The utility of protein and mRNA correlation

    PubMed Central

    Payne, Samuel H.

    2016-01-01

    Transcriptomic, proteomic, and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however, the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight. PMID:25467744

  12. Effects of sodium selenite on aflatoxin B1-induced decrease of ileac T cell and the mRNA contents of IL-2, IL-6, and TNF-? in broilers.

    PubMed

    He, Yang; Fang, Jing; Peng, Xi; Cui, Hengmin; Zuo, Zhicai; Deng, Junliang; Chen, Zhengli; Lai, Weimin; Shu, Gang; Tang, Li

    2014-06-01

    The aim of this work was to assess the protective effect of sodium selenite on the ileum mucosal immunologic toxicity induced by aflatoxin B1 (AFB1). One hundred and eighty one-day-old healthy male avian broilers were divided into four groups of three replicates and 15 birds per replicate and fed with basal diet (control group), 0.3 mg/kg AFB1 (AFB1 group), 0.4 mg/kg Se (+Se group), and 0.3 mg/kg AFB1+0.4 mg/kg Se (AFB1+Se group), respectively. The ileac T-cell subsets were determined by the methods of flow cytometry (FCM), and the mRNA contents of interleukin-2 (IL-2), interleukin-6(IL-6), and tumor necrosis factor-alpha (TNF-?) by quantitative real-time PCR. Compared with those in control group, the percentages of CD3+, CD3+CD4+, CD3+CD8+ intraepithelial lymphocytes (IELs) and LPLs, the CD4+/CD8+ ratio of IELs, and the mRNA contents of IL-2, IL-6, and TNF-? were decreased in AFB1 group. However, compared with those in AFB1 group, these parameters of AFB1+Se group were increased to be close to those in control group. It was concluded that 0.3 mg/kg AFB1 could reduce the cellular immune function of the ileum mucosa, but 0.4 mg/kg supplemented dietary selenium showed protective effects on AFB1-induced immunologic injury. PMID:24807686

  13. Decreased albumin mRNA in immunodeficient wasted' mice

    SciTech Connect

    Libertin, C.R.; Buczek, N.; Weaver, P.; Mobarhan, S.; Woloschak, G.E. Argonne National Lab., IL )

    1991-03-15

    Mice bearing the autosomal recessive gene wst (wst/wst) develop a wasting syndrome' that leads to death by 28-32 days of age. These mice have faulty repair of damage induced by ionizing radiation, immunodeficiency at secretory sites, and neurologic abnormalities. In addition to a progressively more apparent wasted phenotype, wst/wst mice show other features of failure to thrive and malnutrition. Daily body weights of the animals revealed a loss in weight between 25 and 30 days of age, a time during which normal littermates were progressively and rapidly gaining weight. Albumin mRNA levels were measured by dilution dot blot hybridizations of liver-derived RNA preparations from wasted mice, littermates, and parental controls. In all wasted mice, albumin mRNA levels were reduced 5 to 10 fold compared to controls. Northern blots revealed that the albumin mRNA present in wasted mice was normal in length though reduced in amount. These results suggest there may be a relationship between low albumin synthesis and the wasting syndrome of the wst/wst mouse.

  14. Expression of galectin-9 mRNA in hepatocellular carcinoma

    PubMed Central

    Liang, Yong; Chen, Jiayu; Zhang, Yanzi; Zhang, Wei

    2015-01-01

    Liver cancer, with a very high prevalence, is one of the most common causes of death worldwide. Galectin-9, a semi-galactoside-binding protein, was demonstrated to be involved in the formation and metastasis processes of tumors such as breast cancer, and has significant impact on the development and prognosis of tumor. In this study, 90 cases of liver cancer patients who had liver cancer resection surgery treatment, were selected. Samples of liver cancer tissues and cancer-adjacent tissues from the surgery resection of liver cancer patients, which also confirmed by pathology after operation as specimens, were obtained to detect the expression level of Galectin-9 mRNA. The comparing results showed that there were significant differences between the expression of Galectin-9 mRNA in cancer-adjacent tissues and that in cancer tissues (P < 0.05), in terms of pathology differentiation, TNM, and recurrence transfer aspects. However, there were no obvious correlations with gender, age, the size of tumor, and HBsAg. The expression of Galectin-9 mRNA has a close relationship with pathological differentiation, TNM, and recurrence metastasis. Our data presented here provide theoretical basis for new target of liver cancer diagnosis as well as potential prognosis. PMID:26823850

  15. Sequence and regulation of European eel prolactin mRNA.

    PubMed

    Quérat, B; Cardinaud, B; Hardy, A; Vidal, B; D'Angelo, G

    1994-06-01

    cDNA clones encoding the European eel (Anguilla anguilla L.) prolactin were isolated from a pituitary cDNA library constructed in gamma gt10, using a rainbow trout Prl cDNA fragment as a probe. Four different inserts were subcloned into the pGEM 3Z plasmid after PCR amplification. The 1082 bp-long nucleotide sequence revealed an open reading frame of 627 bp encoding a 24 amino acid-long signal peptide followed by a 185 amino acid-long mature protein. Comparison studies showed 60-70% homology with other known teleost fish prolactins and 30-45% with non-teleost fish, amphibian, reptilian, avian and mammalian prolactins. In situ hybridization studies using labelled prolactin RNA probe showed a strong signal in the rostral pars distalis of the pituitary gland. We next examined the physiological regulation of this prolactin synthesis in vivo using Northern blot analysis and prolactin cDNA probe labelled by random priming. The pituitary prolactin mRNA level was markedly decreased 3 weeks after transfer of eels from freshwater to sea water. Implants of thyroid hormones left for up to three weeks were ineffective on prolactin mRNA. Estradiol administered as implant, alone or in combination with 500 micrograms testosterone, was also unable to significantly alter the pituitary mRNA level for prolactin in the freshwater silver eels whatever the dose used (20-500 micrograms) and whatever the duration of treatment (from 4 days to 10 weeks). PMID:7926267

  16. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. )

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  17. Osteonectin mRNA: distribution in normal and transformed cells.

    PubMed Central

    Young, M F; Bolander, M E; Day, A A; Ramis, C I; Robey, P G; Yamada, Y; Termine, J D

    1986-01-01

    Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus. Images PMID:3012473

  18. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  19. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  20. Interleukin 2 receptor-targeted cytotoxicity. Interleukin 2 receptor- mediated action of a diphtheria toxin-related interleukin 2 fusion protein

    PubMed Central

    1988-01-01

    The IL-2 toxin-mediated inhibition of protein synthesis in high affinity IL-2-R-positive murine and human T cell lines has been examined. Both excess free IL-2 and mAb to the Tac epitope of the p55 subunit of IL-2-R are shown to block the action of IL-2 toxin; whereas, agents that interact with other receptors or antigens on the T cell surface have no effect. We show that IL-2 toxin, like diphtheria toxin, must pass through an acidic vesicle in order to intoxicate target T cells. Finally, we demonstrate that the IL-2 toxin-mediated inhibition of protein synthesis in both human and murine T cells that bear the high affinity IL-2-R is due to the classic diphtheria toxin fragment A- catalyzed ADP ribosylation of elongation factor 2. PMID:3126255

  1. Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Takiguchi, Naomi; Suehara, Tetsuya; Takakura, Yoshinobu

    2011-10-01

    Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid-dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA-mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression. PMID:21520018

  2. mRNA decay in spinach chloroplasts: psbA mRNA degradation is initiated by endonucleolytic cleavages within the coding region.

    PubMed Central

    Klaff, P

    1995-01-01

    The expression of chloroplast genes during leaf development in higher plants is regulated on several levels as transcription, RNA processing and stability, protein stability and turnover. Differential mRNA stability is one major component which contributes to the developmentally controlled accumulation of higher plant chloroplast psbA mRNA, which encodes the D1 protein of photosystem II. To understand the molecular mechanisms of specific mRNA degradation an in vitro mRNA decay system based on lysed chloroplasts from spinach leaves was established. Employing this degradation extract the decay of psbA mRNA was analyzed. Half-life of the psbA mRNA in vitro is dependent on the degradation conditions as the presence of Mg2+, which was found to stabilize the mRNA. Addition of tRNA stabilizes the mRNA and allows the accumulation of distinct degradation intermediates. psbA mRNA derived fragments of the same size were detected in degradation experiments in vitro, in organello and in vivo. 5' ends of the degradation intermediates were identified by primer extension and found to be localized in the 5' part of the coding region. The data indicate a degradation mechanism involving initiation of psbA mRNA decay by specific endonucleolytic cleavage and subsequent exonucleolytic degradation of the fragments. Possible models for cleavage site recognition are discussed. Images PMID:8532533

  3. Conceptual Modeling of mRNA Decay Provokes New Hypotheses

    PubMed Central

    Somekh, Judith; Haimovich, Gal; Guterman, Adi; Dori, Dov; Choder, Mordechai

    2014-01-01

    Biologists are required to integrate large amounts of data to construct a working model of the system under investigation. This model is often informal and stored mentally or textually, making it prone to contain undetected inconsistencies, inaccuracies, or even contradictions, not much less than a representation in free natural language. Using Object-Process Methodology (OPM), a formal yet visual and humanly accessible conceptual modeling language, we have created an executable working model of the mRNA decay process in Saccharomyces cerevisiae, as well as the import of its components to the nucleus following mRNA decay. We show how our model, which incorporates knowledge from 43 articles, can reproduce outcomes that match the experimental findings, evaluate hypotheses, and predict new possible outcomes. Moreover, we were able to analyze the effects of the mRNA decay model perturbations related to gene and interaction deletions, and predict the nuclear import of certain decay factors, which we then verified experimentally. In particular, we verified experimentally the hypothesis that Rpb4p, Lsm1p, and Pan2p remain bound to the RNA 3?-untralslated region during the entire process of the 5? to 3? degradation of the RNA open reading frame. The model has also highlighted erroneous hypotheses that indeed were not in line with the experimental outcomes. Beyond the scientific value of these specific findings, this work demonstrates the value of the conceptual model as an in silico vehicle for hypotheses generation and testing, which can reinforce, and often even replace, risky, costlier wet lab experiments. PMID:25255440

  4. The separation of 9 S RNA from avian immature red blood cells into f2c-histone mRNA and globin mRNA.

    PubMed

    Knchel, W; Heyer, I

    1975-07-23

    9 S RNA from avian immature red blood cells was isolated from polysome-released ribonucleoprotein particles by sucrose-gradient techniques. Translation of this RNA in an Ehrlich ascites cell-free system and product analysis revealed that globin mRNA was contaminated by f2c-histone mRNA. When 9 S RNA was applied to oligo(dT)-cellulose columns a partial separation could be achieved. Poly (A)-containing globin mRNA did not contain f2c-histon mRNA, whereas the RNA which was not absorbed to oligo(dT)-cellulose contained all the f2c-histone mRNA besides substantial amounts of globin mRNA. PMID:1148248

  5. Differential regulation of plastid mRNA stability. Progress report

    SciTech Connect

    Stern, D.B.

    1993-09-01

    Our goal is to identify cis-acting sequences and transacting factors that function in plastid mRNA maturation, stabilization, and/or decay through an in vitro and in vivo analysis of mRNA:protein interactions. Our previous results emphasized the study of 3{prime}end inverted repeat sequences (IRs) that serve both as mRNA processing elements and stability determinants, and associate with plastid proteins that potentially play enzymatic, structural and/or regulatory roles. We seek to define, by single base and internal deletion mutagenesis, the sequence and structural requirements for protein binding to the 3{prime} IRs of petD and psbA mRNAs; to purify RNA-binding proteins that demonstrate gene- or sequence-specific binding, or that are implicated in RNA stabilization or decay; and to investigate the native form of mRNA in the plastid, by attempting to purify ribonucleoprotein (RNP) particles from organelles. Our view of mRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNA-binding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with petD 3{prime} IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the petD stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins.

  6. mRNA quality control at the 5' end.

    PubMed

    Zhai, Li-ting; Xiang, Song

    2014-05-01

    All eukaryotic mRNAs are capped at their 5' end. Capping of mRNAs takes place co-transcriptionally and involves three steps. The intermediates of the capping process, as well as the uncapped 5' tri-phosphate RNA, are resistant to decapping and degradation by known factors, leading to the assumption that the capping process always proceeds to completion. This view was recently drastically changed. A novel family of enzymes, including the yeast proteins Rai1, Dxo1/Ydr370C, and the mammalian protein DXO/Dom3Z, has been identified. These enzymes catalyze the conversion of the improperly capped mRNAs to 5' mono-phosphate RNA, allowing them to be degraded by 5'-3' exoribonucleases. Several of these enzymes also possess 5'-3' exoribonuclease activities themselves, and can single-handedly clear the improperly capped mRNAs. Studying of these enzymes has led to the realization that mRNA capping does not always proceed to completion, and the identification of an mRNA capping quality control mechanism in eukaryotes. In this paper, we briefly review recent advances in this area. PMID:24793761

  7. Drosha mediates destabilization of Lin28 mRNA targets

    PubMed Central

    Qiao, Chong; Ma, Jing; Xu, Jie; Xie, Mingyi; Ma, Wei; Huang, Yingqun

    2012-01-01

    Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency. PMID:22935707

  8. Subcellular mRNA localisation at a glance

    PubMed Central

    Parton, Richard M.; Davidson, Alexander; Davis, Ilan; Weil, Timothy T.

    2014-01-01

    ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms. PMID:24833669

  9. Analysis of an mRNA exhibiting anomalous translational specificity.

    PubMed Central

    Vellanoweth, R L; Rabinowitz, J C

    1991-01-01

    Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested. Images PMID:1898927

  10. Regulation of aldose reductase, sorbitol dehydrogenase, and taurine cotransporter mRNA in rat medulla.

    PubMed

    Martial, S; Price, S R; Sands, J M

    1995-05-01

    The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress. PMID:7620095

  11. Mucin1 promotes the migration and invasion of hepatocellular carcinoma cells via JNK-mediated phosphorylation of Smad2 at the C-terminal and linker regions

    PubMed Central

    Wang, Juan; Liu, Guomu; Li, Qiongshu; Wang, Fang; Xie, Fei; Zhai, Ruiping; Guo, Yingying; Chen, Tanxiu; Zhang, Nannan; Ni, Weihua; Yuan, Hongyan; Tai, Guixiang

    2015-01-01

    Mucin1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas. In this study, wound-healing, transwell migration and matrigel invasion assays showed that MUC1 promotes human hepatocellular carcinoma (HCC) cell migration and invasion by MUC1 gene silencing and overexpressing. Treatment with exogenous transforming growth factor beta (TGF-β)1, TGF-β type I receptor (TβRI) inhibitor, TGF-β1 siRNAs, or activator protein 1 (AP-1) inhibitor to MUC1-overexpressing HCC cells revealed that MUC1-induced autocrine TGF-β via JNK/AP-1 pathway promotes the cell migration and invasion. In addition, the migration and invasion of HCC cells were more significantly inhibited by JNK inhibitor compared with that by TβRI inhibitor or TGF-β1 siRNAs. Further studies demonstrated that MUC1-mediated JNK activation not only enhances the phosphorylation of Smad2 C-terminal at Ser-465/467 site (Smad2C) through TGF-β/TβRI, but also directly enhances the phosphorylation of Smad2 linker region at Ser-245/250/255 site (Smad2L), and then both of them collaborate to upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of HCC. These results indicate that MUC1 is an attractive target in liver cancer therapy. PMID:26057631

  12. Retinoic acid influences the embryoid body formation in mouse embryonic stem cells by induction of caspase and p38 MAPK/JNK-mediated apoptosis.

    PubMed

    Huang, Fu-Jen; Lan, Kuo-Chung; Kang, Hong-Yo; Lin, Pin-Yao; Chan, Wen-Hsiung; Hsu, Yu-Cheng; Liu, Yen-Chih; Huang, Ko-En

    2013-04-01

    Although all-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development, high levels are teratogenic in many species. RA results in immediate effects on the preimplantation embryo and on blastocyst development in vitro and in vivo. To further elucidate the cellular mechanisms of early postimplantation embryo development induced by RA, we present an embryonic cell line, B5, as a candidate system for the investigation of these processes. We used undifferentiated ES cells as the model, which is from the undifferentiated status to differentiated status [embryoid body (EB) formation] mimicking postimplantation embryo development (egg-cylinder stage of embryo formation) to clarify the cellular mechanism of action of RA in the implanted blastocysts and cell apoptosis following the series of exposures to differing RA concentrations. Using an in vitro model, we identified the impact of RA on undifferentiated embryonic stem (ES) cells, including inhibition of cell proliferation and induction of cell apoptosis. JNK, P-38 and caspase activation were shown in the nature of RA-triggered apoptotic signaling in ES cells. The carry-on influences of RA on the ES cell were shown in the formation of EB from the pretreated ES cells. RA resulted in apparent impact on undifferentiated ES cells in vitro, with increased numbers of apoptotic cells initially and inhibited cell proliferation, which led to decreased size of EB. The process of EB formation (mimicking the early postimplantation embryo development) is regulated by RA-induced apoptosis through the activation of caspase and P38 MAPK/JNK pathway. PMID:21626648

  13. Visualizing mRNA Dynamics in Live Neurons and Brain Tissues.

    PubMed

    Park, Hye Yoon; Song, Minho

    2016-01-01

    Localization of mRNA plays a crucial role in a variety of neuronal processes including synaptogenesis, axonal guidance, and long-term plasticity. Recent advances in fluorescence imaging and RNA labeling techniques allow us to visualize how individual mRNA molecules are dynamically regulated inside live neurons and brain tissues. Here, we describe key methods in imaging mRNA dynamics, including preparation of neuron culture and brain slices from transgenic mice expressing GFP-labeled mRNA, high-resolution detection of single molecules, live tissue imaging, and analysis of mRNA transport. PMID:26463394

  14. Corrections for mRNA extraction and sample normalization errors find increased mRNA levels may compensate for cancer haplo-insufficiency

    PubMed Central

    Weaver, David A; Nestor-Kalinoski, Andrea L; Craig, Kristen; Gorris, Matthew; Parikh, Tejal; Mabry, Helen; Allison, David C

    2014-01-01

    The relative mRNA levels of differentially expressed (DE) and housekeeping (HK) genes of six aneuploid cancer lines with large-scale genomic changes identified by SNP/SKY analysis were compared with similar genes in diploid cells. The aneuploid cancer lines had heterogeneous genomic landscapes with subdiploid, diploid, and supradiploid regions and higher overall gene copy numbers compared with diploid cells. The mRNA levels of the haploid, diploid, and triploid HK genes were found to be higher after correction of easily identifiable mRNA measurement errors. Surprisingly, diploid and aneuploid HK gene mRNA levels were the same by standard expression array analyses, despite the higher copy numbers of the cancer cell HK genes. This paradoxical result proved to be due to inaccurate inputs of true intra-cellular mRNAs for analysis. These errors were corrected by analyzing the expression intensities of DE and HK genes in mRNAs extracted from equal cell numbers (50:50) of intact cancer cell and lymphocyte mixtures. Correction for both mRNA extraction/sample normalization errors and total gene copy numbers found the SUIT-2 and PC-3 cell lines' cancer genes both had ?50% higher mRNA levels per single allele than lymphocyte gene alleles. These increased mRNA levels for single transcribed cancer alleles may restore functional mRNA levels to cancer genes rendered haplo-insufficient by the genetic instability of cancer. 2013 Wiley Periodicals, Inc. PMID:24327546

  15. Interrelations between translation and general mRNA degradation in yeast.

    PubMed

    Huch, Susanne; Nissan, Tracy

    2014-01-01

    Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. PMID:24944158

  16. Ratcheting mRNA out of the nucleus.

    PubMed

    Stewart, Murray

    2007-02-01

    Export of mature mRNA to the cytoplasm is the culmination of the nuclear portion of eukaryotic gene expression. After transport-competent mature mRNP export complexes are formed in the nucleus, their passage through nuclear pore complexes (NPCs) is facilitated by the Mex67:Mtr2 heterodimer. At the NPC cytoplasmic face, mRNP remodeling prevents its return to the nucleus and so functions as a molecular ratchet imposing directionality on transport. In budding yeast, recent work suggests that the DEAD-box helicase Dbp5 remodels mRNPs at the NPC cytoplasmic face by removing Mex67 and that the Dbp5 ATPase is activated by Gle1 and inositol hexaphosphate (IP(6)). PMID:17289581

  17. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain. PMID:24161824

  18. Origin of hepatitis delta virus mRNA.

    PubMed

    Gudima, S; Wu, S Y; Chiang, C M; Moraleda, G; Taylor, J

    2000-08-01

    Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5' end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5'-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5' ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5' end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3'-end addition to the template. These new findings support the interpretation that the 5' end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping. PMID:10906174

  19. Analyzing Subcellular mRNA Localization via Cell Fractionation

    PubMed Central

    Jagannathan, Sujatha; Nwosu, Christine; Nicchitta, Christopher V.

    2013-01-01

    Summary The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon. The partitioning of mRNAs to the ER is generally thought to be achieved by the signal recognition particle (SRP) pathway. In this pathway, mRNA localization to the ER is determined by the translation product – translation yields an N-terminal signal sequence or topogenic signal that is recognized by the SRP and the resulting mRNA-ribosome-SRP complex is then recruited to the ER membrane. Recent studies have demonstrated that mRNAs can be localized to the ER via a signal sequence and/or translation-independent pathway(s) and that discrete sets of cytosolic protein-encoding mRNAs are enriched on the ER membrane, though they lack an encoded signal sequence. These key findings reopen investigations into the mechanism(s) that govern mRNA localization to the ER. In this contribution, we describe two independent methods that can be utilized to study this important and poorly understood aspect of eukaryotic cell biology. These methods comprise two independent means of fractionating tissue culture cells to yield free/cytosolic polyribosomes and ER membrane-bound polyribosomes. Detailed methods for the fractionation and characterization of the two polyribosome pools are provided. PMID:21431749

  20. Interleukin-2 receptor downstream events in regulatory T cells

    PubMed Central

    Zeiser, Robert; Negrin, Robert S.

    2010-01-01

    Naturally occurring CD4+CD25highFOXP3+ regulatory T cells (Tregs) constitute a powerful mechanism of immune regulation and therefore, have important therapeutic potential for disorders such as autoimmune diseases, allograft rejection and graft-versus-host disease. Disruption of the IL-2R signalling pathway by genetic defects of the interleukin (IL)-2 gene or components of the IL-2 receptor (R) complex results in severe T cell-mediated autoimmunity rather than immunodeficiency, indicating a crucial role for IL-2R signalling for Treg development and function. Signalling downstream of the IL-2R can act through the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway, the Janus kinase (JAK)/Signal transducers and Activators of Transcription (STAT) pathway and the mitogen-activated protein kinase (MAPK) pathway. In this report we focus on the relevance of these pathways as well as the impact of immunosuppressive drugs that may affect or enhance Treg function by targeting IL-2R signalling. PMID:18235249

  1. Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

    PubMed

    Roth, C; Mir, L M; Cressent, M; Quintin-Colonna, F; Ley, V; Fradelizi, D; Kourilsky, P

    1992-12-01

    Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo. PMID:1286066

  2. Rituximab Plus Interleukin-2 in Treating Patients With Hematologic Cancer

    ClinicalTrials.gov

    2013-06-05

    B-cell Adult Acute Lymphoblastic Leukemia; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Noncontiguous Stage II Adult Burkitt Lymphoma; Noncontiguous Stage II Adult Diffuse Large Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Mixed Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Small Cleaved Cell Lymphoma; Noncontiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Noncontiguous Stage II Adult Lymphoblastic Lymphoma; Noncontiguous Stage II Grade 1 Follicular Lymphoma; Noncontiguous Stage II Grade 2 Follicular Lymphoma; Noncontiguous Stage II Grade 3 Follicular Lymphoma; Noncontiguous Stage II Mantle Cell Lymphoma; Noncontiguous Stage II Marginal Zone Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic 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 Small Lymphocytic Lymphoma; Splenic Marginal Zone Lymphoma; Stage III Adult Burkitt Lymphoma; Stage III Adult Diffuse Large Cell Lymphoma; Stage III Adult Diffuse Mixed Cell Lymphoma; Stage III Adult Diffuse Small Cleaved Cell Lymphoma; Stage III Adult Immunoblastic Large Cell Lymphoma; Stage III Adult Lymphoblastic Lymphoma; Stage III Grade 1 Follicular Lymphoma; Stage III Grade 2 Follicular Lymphoma; Stage III Grade 3 Follicular Lymphoma; Stage III Mantle Cell Lymphoma; Stage III Marginal Zone Lymphoma; Stage III Small Lymphocytic Lymphoma; Stage IV Adult Burkitt Lymphoma; Stage IV Adult Diffuse Large Cell Lymphoma; Stage IV Adult Diffuse Mixed Cell Lymphoma; Stage IV Adult Diffuse Small Cleaved Cell Lymphoma; Stage IV Adult Immunoblastic Large Cell Lymphoma; Stage IV Adult Lymphoblastic Lymphoma; Stage IV Grade 1 Follicular Lymphoma; Stage IV Grade 2 Follicular Lymphoma; Stage IV Grade 3 Follicular Lymphoma; Stage IV Mantle Cell Lymphoma; Stage IV Marginal Zone Lymphoma; Stage IV Small Lymphocytic Lymphoma

  3. Interleukin-2 gene therapy of surgical minimal residual tumour disease.

    PubMed

    Vlk, V; Rssner, P; Indrov, M; Bubenk, J; Sobota, V

    1998-03-30

    Our study was designed to examine the effects of IL-2 gene therapy in a surgical minimal residual tumour disease (SMRTD). Mice were inoculated s.c. with methylcholanthrene (MC)-induced MC12 sarcoma cells. When the tumours reached 8 to 12 mm in diameter, they were excised, either completely ("microscopic SMRTD") or incompletely ("macroscopic SMRTD"). On day 90 after surgery, the tumour recurrence rate in untreated mice with microscopic SMRTD was approximately 30%, whereas in those with macroscopic SMRTD it was 75%. After surgery, experimental mice were treated with 2 types of irradiated, IL-2 gene-modified, IL-2-producing tumour cell vaccine. One type of vaccine was derived from the MC12 sarcoma cells (MC12-1L2/IV-3); the other type was derived from an unrelated X63-Ag8.653 plasmacytoma (X63-m-IL-2). Both types of vaccine failed to cure the macroscopic SMRTD. Whereas the X63-m-IL-2 vaccine was also ineffective in the microscopic SMRTD, the MC12-IL2/IV-3 vaccine was capable of preventing growth in all but one mouse (1164) with microscopic SMRTD when administered 2 to 5 days after surgery. If the vaccination took place 2 days before surgery or later than 5 days after surgery, the therapeutic activity was lost. Vaccination with irradiated parental MC12 cells did not produce any significant benefit compared to the operated-only mice. The protective effect of the MC12-L2/IV-3 vaccine was specific and comparatively long-lasting. Vaccinated mice, which had rejected the MC12 tumour residuum, were capable of rejecting a second inoculum of the MC12 sarcoma cells injected on days 35 to 110 after surgery but succumbed to the growth of 2 other unrelated murine sarcomas carrying different tumour-rejection antigens. PMID:9533770

  4. Increased polyamines may downregulate interleukin 2 production in rheumatoid arthritis.

    PubMed Central

    Flescher, E; Bowlin, T L; Ballester, A; Houk, R; Talal, N

    1989-01-01

    Polyamines downregulate immune reactivity. RA is associated with decreased IL 2 production. In this study, we present evidence to suggest that excessive polyamines can contribute to the IL 2 deficiency in RA. Blocking polyamine production with inhibitors of ornithine decarboxylase results in increased IL 2 production by RA PBMC. Moreover, polyamine oxidase (PAO) inhibitors and catalase also increase IL 2 production by RA PBMC. This effect of PAO inhibition is monocyte mediated. After 3 d in culture, RA PBMC produce three times more IL 2 than do normal PBMC. This rise is prevented by exogenous spermidine but only in the presence of monocytes. The concentration of polyamines in RA PBMC and synovial fluid MNC is 2-20-fold higher than in normal cells. Thus, polyamines and their oxidation products downregulate IL 2 production by RA PBMC and may account for the decreased T cell effector function seen in this disease. PMID:2784801

  5. Lymphocyte Subsets and Interleukin-2 Receptors in Autistic Children.

    ERIC Educational Resources Information Center

    Denney, Douglas R.; And Others

    1996-01-01

    Blood samples were obtained from 10 male autistic children, ages 7-15 years, and 10 controls. The children with autism had a lower percentage of helper-inducer cells and a lower helper:suppressor ratio, with both measures inversely related to the severity of autistic symptoms. (Author/DB)

  6. An agent-based model for mRNA export through the nuclear pore complex

    PubMed Central

    Azimi, Mohammad; Bulat, Evgeny; Weis, Karsten; Mofrad, Mohammad R. K.

    2014-01-01

    mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics. PMID:25253717

  7. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  8. Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C

    PubMed Central

    Kasprzak, Aldona; Adamek, Agnieszka; Przybyszewska, Wies?awa; Pyda, Przemys?aw; Szmeja, Jacek; Seraszek-Jaros, Agnieszka; Lanzafame, Agata; Surdacka, Anna; Mozer-Lisewska, Iwona; Koczorowska, Maria

    2015-01-01

    AIM: To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturers instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile. PMID:25852271

  9. Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3?UTR

    PubMed Central

    Kim, Min Young; Park, Jungyun; Lee, Jong Joo; Ha, Dae Hyun; Kim, Jonghwan; Kim, Chan Gil; Hwang, Jungwook; Kim, Chul Geun

    2014-01-01

    Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3? untranslated region (3?UTR), referred to as G8, was overexpressed in K562 cells, ?-globin expression was induced, suggesting that the 3?UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3?UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNAligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3?UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stemloop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD. PMID:24799437

  10. Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli

    PubMed Central

    Geyer, Chelsie N.; Fowler, Randal C.; Johnson, James R.; Johnston, Brian; Weissman, Scott J.; Hawkey, Peter; Hanson, Nancy D.

    2016-01-01

    Objectives High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. Methods Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT–PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. Results mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2–48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5–15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2–3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm. Conclusions CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels. PMID:26612874

  11. Intracellular Calcium Regulates Nonsense-Mediated mRNA Decay

    PubMed Central

    Nickless, Andrew; Jackson, Erin; Marasa, Jayne; Nugent, Patrick; Mercer, Robert W.; Piwnica-Worms, David; You, Zhongsheng

    2014-01-01

    The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons (PTCs) and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent an important target for therapeutic intervention. Here we have developed a novel multicolored, bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides including ouabain and digoxin as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the Na+/K+-ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has important implications for exploiting NMD in the treatment of disease. PMID:25064126

  12. Regulation of mRNA translation during mitosis

    PubMed Central

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-01-01

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ?200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function. DOI: http://dx.doi.org/10.7554/eLife.07957.001 PMID:26305499

  13. Molecular Determinants of the Axonal mRNA Transcriptome

    PubMed Central

    Gomes, Cynthia; Merianda, Tanuja T.; Lee, Seung Joon; Yoo, Soonmoon; Twiss, Jeffery L.

    2014-01-01

    Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in cell body responses to axotomy. Recent studies have begun to identify the protein products that contribute to these autonomous responses of axons. In the peripheral nervous system, intra-axonal protein synthesis has been implicated in the localized in vivo responses to neuropathic stimuli, and there is emerging evidence for protein synthesis in CNS axons in vivo. Despite that hundreds of mRNAs have now been shown to localize into the axonal compartment, knowledge of what RNA binding proteins are responsible for this is quite limited. Here, we review the current state of knowledge of RNA transport mechanisms, and highlight recently uncovered mechanisms for dynamically altering the axonal transcriptome. Both changes in the levels or activities of components of the RNA transport apparatus and alterations in transcription of transported mRNAs can effectively shift the axonal mRNA population. Consistent with this, the axonal RNA population shifts with development, with changes in growth state, and in response to extracellular stimulation. Each of these events must impact the transcriptional and transport apparatuses of the neuron, thus directly and indirectly modifying the axonal transcriptome. PMID:23959706

  14. Intronic hammerhead ribozymes in mRNA biogenesis.

    PubMed

    Garca-Robles, Inmaculada; Snchez-Navarro, Jess; de la Pea, Marcos

    2012-11-01

    Small self-cleaving ribozymes are a group of natural RNAs that are capable of catalyzing their own and sequence-specific endonucleolytic cleavage. One of the most studied members is the hammerhead ribozyme (HHR), a catalytic RNA originally discovered in subviral plant pathogens but recently shown to reside in a myriad of genomes along the tree of life. In eukaryotes, most of the genomic HHRs seem to be related to short interspersed retroelements, with the main exception of a group of strikingly conserved ribozymes found in the genomes of all amniotes (reptiles, birds and mammals). These amniota HHRs occur in the introns of a few specific genes, and clearly point to a preserved biological role during pre-mRNA biosynthesis. More specifically, bioinformatic analysis suggests that these intronic ribozymes could offer a new form of splicing regulation of the mRNA of higher vertebrates. We review here the latest advances in the discovery and biological characterization of intronic HHRs of vertebrates, including new conserved examples in the genomes of the primitive turtle and coelacanth fish. PMID:23109545

  15. mRNA related to insulin family in human placenta

    SciTech Connect

    Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  16. Oscillatory kinetics of gene expression: Protein conversion and slow mRNA transport

    SciTech Connect

    Zhdanov, V. P.

    2009-06-15

    The negative feedback between mRNA and regulatory-protein production may result in oscillations in the kinetics of gene expression if the mRNA-protein interplay includes protein conversion. Using a mean-field kinetic model, we show that such oscillations can be amplified due to limitations of the mRNA transport between the nucleus and cytoplasm. This effect may be dramatic for the mRNA population in the nucleus.

  17. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

    PubMed Central

    Dreyfuss, G; Adam, S A; Choi, Y D

    1984-01-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo. Images PMID:6717428

  18. Differential stability of mRNA species of Alcaligenes eutrophus soluble and particulate hydrogenases.

    PubMed Central

    Oelmüller, U; Schlegel, H G; Friedrich, C G

    1990-01-01

    The functional half-lives of Alcaligenes eutrophus hydrogenase mRNAs were determined by physiological studies. Evidence was obtained for a functional half-life of about 1 h for the soluble NAD-linked hydrogenase (HoxS) mRNA and 14 min for the particulate hydrogenase (HoxP) mRNA. The synthesis of active HoxS continued for about 4 h, albeit at a decreasing rate after inhibition of transcription, e.g., by rifampin. In this strain, the mRNA of HoxS appeared to be stable, while the mRNA of HoxP did not. Different species of hoxS mRNA were detected by the Northern (RNA) hybridization technique using as a probe plasmid pCH139 carrying hoxS structural genes. The sizes of the major hoxS mRNA species were 7.6, 6.2, 5.0, and 0.9 kb. The chemical half-lives of these species ranged from 1 h (5.0-kb mRNA) to 7 h (0.9-kb mRNA). Evidence for a specific cleavage of the 6.2-kb transcript yielding the 0.9-kb species was obtained from RNA-DNA hybridizations with subcloned hoxS DNA. The chemical half-life of total hoxP mRNA was 8 min. Images PMID:1701427

  19. Postnatal development of galanin-like peptide mRNA expression in rat hypothalamus.

    PubMed

    Kawagoe, Rinko; Yamamoto, Yukiyo; Kubo, Kazuyasu; Dobashi, Kazushige; Asayama, Kohtaro; Ueta, Yoichi; Shirahata, Akira

    2008-01-10

    We examined the developmental change of GALP mRNA in male and female rat hypothalamus during postnatal day 1 to 60, using in situ hybridization histochemistry. Neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA in the hypothalamus were also examined because they are important in the regulation of food intake. GALP mRNA was first detected in the arcuate nucleus (ARC) on day 8. GALP mRNA was gradually increased between day 8 and 14 and markedly increased between day 14 and 40, which is the weaning and pubertal period in rats. After day 40, there were no significant differences in GALP mRNA. In contrast to GALP, NPY and POMC mRNAs were detected in the ARC from day 1 and lasted to day 60. There was no sexual dimorphism in GALP, NPY and POMC mRNAs during postnatal development. Next, we examined the effect of the milk deprivation for 24 h on GALP, NPY and POMC mRNA in pups. GALP mRNA did not change by milk deprivation on day 9 and 15, while milk deprivation had a significant effect on NPY and POMC mRNA on day 15. These results suggest that the development of GALP may be associated with developmental changes such as weaning, feeding and maturation of reproductive functions. The regulatory mechanism of GALP mRNA is different from that of the NPY and POMC genes during postnatal development. PMID:17950941

  20. A new function of glucocorticoid receptor: regulation of mRNA stability

    PubMed Central

    Park, Ok Hyun; Do, Eunjin; Kim, Yoon Ki

    2015-01-01

    It has long been thought that glucocorticoid receptor (GR) functions as a DNA-binding transcription factor in response to its ligand (a glucocorticoid) and thus regulates various cellular and physiological processes. It is also known that GR can bind not only to DNA but also to mRNA; this observation points to the possible role of GR in mRNA metabolism. Recent data revealed a molecular mechanism by which binding of GR to target mRNA elicits rapid mRNA degradation. GR binds to specific RNA sequences regardless of the presence of a ligand. In the presence of a ligand, however, the mRNA-associated GR can recruit PNRC2 and UPF1, both of which are specific factors involved in nonsense-mediated mRNA decay (NMD). PNRC2 then recruits the decapping complex, consequently promoting mRNA degradation. This mode of mRNA decay is termed GR-mediated mRNA decay (GMD). Further research demonstrated that GMD plays a critical role in chemotaxis of immune cells by targeting CCL2 mRNA. All these observations provide molecular insights into a previously unappreciated function of GR in posttranscriptional regulation of gene expression. [BMB Reports 2015; 48(7): 367-368] PMID:26169194

  1. Coupling mRNA processing with transcription in time and space.

    PubMed

    Bentley, David L

    2014-03-01

    Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3' end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes. PMID:24514444

  2. TOPICAL REVIEW: Mechanisms governing the control of mRNA translation

    NASA Astrophysics Data System (ADS)

    Livingstone, Mark; Atas, Evrim; Meller, Amit; Sonenberg, Nahum

    2010-06-01

    The translation of cellular mRNA to protein is a tightly controlled process often deregulated in diseases such as cancer. Furthering our understanding of mRNA structural elements and the intracellular proteins and signaling pathways that affect protein expression is crucial in the development of new therapies. In this review, we discuss the current state-of-the-art of detecting and determining the role of mRNA sequence elements in regulating the initiation of mRNA translation and the therapeutic strategies that exploit this knowledge to treat disease.

  3. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-? -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing

    NASA Astrophysics Data System (ADS)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

    In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human ? -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and ? -globin sequences, we show that the NS1 5' splice site was effectively utilized by the ? -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the ? -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from ? -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

  4. Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3' end of the mRNA.

    PubMed Central

    Levine, B J; Chodchoy, N; Marzluff, W F; Skoultchi, A I

    1987-01-01

    The role of the 3' end of mRNA in coupling between the level of histone mRNAs and DNA synthesis was examined. We introduced modified mouse histone H3 genes into mouse fibroblasts and studied the regulation of several different H3 mRNAs that are not terminated with a normal histone 3' end. In two cases, the stem-loop sequences were deleted from the mRNAs and replaced either by 3' sequences flanking the H3 gene or by globin 3' untranslated region sequences including the polyadenylylation signal. In the former case, approximately equal to 50% of the modified mRNA was polyadenylylated, whereas in the latter case all of the mRNA had a polyadenylylated terminus. In contrast to the normal histone mRNAs, these mRNAs, including the nonadenylylated form, were stable when DNA synthesis was inhibited with several drugs. The levels of two other histone mRNAs, each containing the stem-loop sequences as an internal part of the mRNA, also were stable when DNA synthesis was inhibited. These results indicate that the posttranscriptional coupling of histone mRNA levels to DNA synthesis requires the presence of the stem-loop sequences at the 3' end of the mRNA. Images PMID:2888112

  5. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    PubMed Central

    Blair, E D; Blair, C C; Wagner, E K

    1987-01-01

    To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation. Images PMID:3037112

  6. Exercise and adrenaline increase PGC-1? mRNA expression in rat adipose tissue

    PubMed Central

    Sutherland, Lindsey N; Bomhof, Marc R; Capozzi, Lauren C; Basaraba, Susan A U; Wright, David C

    2009-01-01

    The purpose of the present investigation was to explore the effects of exercise and adrenaline on the mRNA expression of PGC-1?, a master regulator of mitochondrial biogenesis, in rat abdominal adipose tissue. We hypothesized that (1) exercise training would increase PGC-1? mRNA expression in association with increases in mitochondrial marker enzymes, (2) adrenaline would increase PGC-1? mRNA expression and (3) the effect of exercise on PGC-1? mRNA expression in white adipose tissue would be attenuated by a ?-blocker. Two hours of daily swim training for 4 weeks led to increases in mitochondrial marker proteins and PGC-1? mRNA expression in epididymal and retroperitoneal fat depots. Additionally, a single 2 h bout of exercise led to increases in PGC-1? mRNA expression immediately following exercise cessation. Adrenaline treatment of adipose tissue organ cultures led to dose-dependent increases in PGC-1? mRNA expression. A supra-physiological concentration of adrenaline increased PGC-1? mRNA expression in epididymal but not retroperitoneal adipose tissue. ?-Blockade attenuated the effects of an acute bout of exercise on PGC-1? mRNA expression in epididymal but not retroperitoneal fat pads. In summary, this is the first investigation to demonstrate that exercise training, an acute bout of exercise and adrenaline all increase PGC-1? mRNA expression in rat white adipose tissue. Furthermore it would appear that increases in circulating catecholamine levels may be one potential mechanism mediating exercise induced increases in PGC-1? mRNA expression in rat abdominal adipose tissue. PMID:19221126

  7. Interrelations between translation and general mRNA degradation in yeast

    PubMed Central

    Huch, Susanne; Nissan, Tracy

    2014-01-01

    Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747763. doi: 10.1002/wrna.1244 PMID:24944158

  8. Distinguishing direct from indirect roles for bicoid mRNA localization factors

    PubMed Central

    Weil, Timothy T.; Xanthakis, Despina; Parton, Richard; Dobbie, Ian; Rabouille, Catherine; Gavis, Elizabeth R.; Davis, Ilan

    2010-01-01

    Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. Here we use live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. We show that Swallow and bicoid mRNA are transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, and we present evidence that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. Our results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead we propose a model for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport. PMID:20023172

  9. RELATION OF MRNA REVERSE TRANSCRIPTASE-PCR SIGNAL TO CAMPYLOBACTER SPP. COLONIZATION OF CHICKS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chicken colonization by cells of Campylobacter jejuni having positive mRNA Reverse Transcriptase-PCR (RT-PCR) signal, but which are non-cultivable, would provide a means to relate cell viability with mRNA signal. In addition, the role of viable but non-cultivable (VBNC) forms of Campylobacter spp. f...

  10. Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA.

    PubMed

    Daz-Toledano, Rosa; Gmez, Jordi

    2015-10-01

    The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself. PMID:25900662

  11. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function

    PubMed Central

    Fedyunin, Ivan; Ignatova, Zoya

    2015-01-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. PMID:26495981

  12. Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.

    PubMed

    Schmidt, Yvonne; Biniossek, Martin; Stark, G Bjrn; Finkenzeller, Gnter; Simunovic, Filip

    2015-03-01

    Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications. PMID:25536665

  13. In vitro synthesis of modified mRNA for induction of protein expression in human cells.

    PubMed

    Avci-Adali, Meltem; Behring, Andreas; Steinle, Heidrun; Keller, Timea; Krajeweski, Stefanie; Schlensak, Christian; Wendel, Hans P

    2014-01-01

    The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express. PMID:25489992

  14. Disrupted-in-schizophrenia 1 regulates transport of ITPR1 mRNA for synaptic plasticity.

    PubMed

    Tsuboi, Daisuke; Kuroda, Keisuke; Tanaka, Motoki; Namba, Takashi; Iizuka, Yukihiko; Taya, Shinichiro; Shinoda, Tomoyasu; Hikita, Takao; Muraoka, Shinsuke; Iizuka, Michiro; Nimura, Ai; Mizoguchi, Akira; Shiina, Nobuyuki; Sokabe, Masahiro; Okano, Hideyuki; Mikoshiba, Katsuhiko; Kaibuchi, Kozo

    2015-05-01

    Disrupted-in-schizophrenia 1 (DISC1) is a susceptibility gene for major psychiatric disorders, including schizophrenia. DISC1 has been implicated in neurodevelopment in relation to scaffolding signal complexes. Here we used proteomic analysis to screen for DISC1 interactors and identified several RNA-binding proteins, such as hematopoietic zinc finger (HZF), that act as components of RNA-transporting granules. HZF participates in the mRNA localization of inositol-1,4,5-trisphosphate receptor type 1 (ITPR1), which plays a key role in synaptic plasticity. DISC1 colocalizes with HZF and ITPR1 mRNA in hippocampal dendrites and directly associates with neuronal mRNAs, including ITPR1 mRNA. The binding potential of DISC1 for ITPR1 mRNA is facilitated by HZF. Studies of Disc1-knockout mice have revealed that DISC1 regulates the dendritic transport of Itpr1 mRNA by directly interacting with its mRNA. The DISC1-mediated mRNA regulation is involved in synaptic plasticity. We show that DISC1 binds ITPR1 mRNA with HZF, thereby regulating its dendritic transport for synaptic plasticity. PMID:25821909

  15. Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

    PubMed

    Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

    2014-12-23

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

  16. Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

  17. Axonal Amphoterin mRNA Is Regulated by Translational Control and Enhances Axon Outgrowth

    PubMed Central

    Merianda, Tanuja T.; Coleman, Jennifer; Kim, Hak Hee; Kumar Sahoo, Pabitra; Gomes, Cynthia; Brito-Vargas, Paul; Rauvala, Heikki; Blesch, Armin; Yoo, Soonmoon

    2015-01-01

    High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3?-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3?-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3?-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body. PMID:25855182

  18. Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

    PubMed Central

    2015-01-01

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

  19. Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA.

    PubMed

    Forget, Amlie; Chartrand, Pascal

    2011-03-01

    Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA. PMID:21468235

  20. Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA

    PubMed Central

    Forget, Amlie

    2011-01-01

    Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA. PMID:21468235

  1. PROLONGED FASTING AND CORTISOL REDUCE MYOSTATIN MRNA LEVELS IN TILAPIA LARVAE, SHORT-TERM FASTING ELEVATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indices in adults, skeletal muscle myostatin mRNA...

  2. Towards Targeted Delivery Systems: Ligand Conjugation Strategies for mRNA Nanoparticle Tumor Vaccines

    PubMed Central

    Phua, Kyle K. L.

    2015-01-01

    The use of nanoparticles encapsulating messenger RNA (mRNA) as a vaccine has recently attracted much attention because of encouraging results achieved in many nonviral genetic antitumor vaccination studies. Notably, in all of these studies, mRNA nanoparticles are passively targeted to dendritic cells (DCs) through careful selection of vaccination sites. Hence, DC-targeted mRNA nanoparticle vaccines may be an imminent next step forward. In this brief report, we will discuss established conjugation strategies that have been successfully applied to both polymeric and liposomal gene delivery systems. We will also briefly describe promising DC surface receptors amenable for targeting mRNA nanoparticles. Practicable conjugation strategies and receptors reviewed in this paper will provide a convenient reference to facilitate future development of targeted mRNA nanoparticle vaccine. PMID:26819957

  3. In the right place at the right time: visualizing and understanding mRNA localization.

    PubMed

    Buxbaum, Adina R; Haimovich, Gal; Singer, Robert H

    2015-02-01

    The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization. PMID:25549890

  4. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells.

    PubMed

    Shi, Jing; Zhou, Ming; Gong, Aihua; Li, Qijun; Wu, Qian; Cheng, Gary J; Yang, Mingyang; Sun, Yaocheng

    2016-02-16

    The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biological and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepared nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomolecules. PMID:26813157

  5. In the right place at the right time: visualizing and understanding mRNA localization

    PubMed Central

    Buxbaum, Adina R.; Haimovich, Gal

    2015-01-01

    The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization. PMID:25549890

  6. Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles.

    PubMed

    Baba, Miyuki; Itaka, Keiji; Kondo, Kenji; Yamasoba, Tatsuya; Kataoka, Kazunori

    2015-03-10

    Sensory nerve disorders are difficult to cure completely considering poor nerve regeneration capacity and difficulties in accurately targeting neural tissues. Administering mRNA is a promising approach for treating neurological disorders because mRNA can provide proteins and peptides in their native forms for mature non-dividing neural cells, without the need of entering their nuclei. However, direct mRNA administration into neural tissues in vivo has been challenging due to too unstable manner of mRNA and its strong immunogenicity. Thus, using a suitable carrier is essential for effective mRNA administration. For this purpose, we established a novel carrier based on the self-assembly of polyethylene glycol (PEG)-polyamino acid block copolymer, i.e. polyplex nanomicelles. To investigate the feasibility and efficacy of mRNA administration for the treatment of sensory nerve disorders, we used a mouse model of experimentally induced olfactory dysfunction. Intranasal administration of mRNA-loaded nanomicelles provided an efficient and sustained protein expression for nearly two days in nasal tissues, particularly in the lamina propria which contains olfactory nerve fibers, with effectively regulating the immunogenicity of mRNA. Consequently, once-daily intranasal administration of brain-derived neurotrophic factor (BDNF)-expressing mRNA using polyplex nanomicelles remarkably enhanced the neurological recovery of olfactory function along with repairing the olfactory epithelium to a nearly normal architecture. To the best of our knowledge, this is the first study to show the therapeutic potential of introducing exogenous mRNA for the treatment of neurological disorders. These results indicate the feasibility and safety of using mRNA, and provide a novel strategy of mRNA-based therapy. PMID:25599855

  7. Comparison of Protamine 1 to Protamine 2 mRNA Ratio and YBX2 gene mRNA Content in Testicular Tissue of Fertile and Azoospermic Men

    PubMed Central

    Moghbelinejad, Sahar; Najafipour, Reza; Hashjin, Amir Samimi

    2015-01-01

    Background Although aberrant protamine (PRM) ratios have been observed in infertile men, the mechanisms that implicit the uncoupling of PRM1 and PRM2 expression remain unclear. To uncover these mechanisms, in this observational study we have compared the PRM1/PRM2 mRNA ratio and mRNA contents of two regulatory factors of these genes. Materials and Methods In this experimental study, sampling was performed by a multi-step method from 50 non-obstructive azoospermic and 12 normal men. After RNA extraction and cDNA synthesis, real-time quantitative polymerase chain reaction (RT- QPCR) was used to analyze the PRM1, PRM2, Y box binding protein 2 (YBX2) and JmjC-containing histone demethylase 2a (JHDM2A) genes in testicular biopsies of the studied samples. Results The PRM1/PRM2 mRNA ratio differed significantly among studied groups, namely 0.21 0.13 in azoospermic samples and -0.8 0.22 in fertile samples. The amount of PRM2 mRNA, significantly reduced in azoospermic patients. Azoospermic men exhibited significant under expression of YBX2 gene compared to controls (P<0.001). mRNA content of this gene showed a positive correlation with PRM mRNA ratio (R=0.6, P=0.007). JHDM2A gene expression ratio did not show any significant difference between the studied groups (P=0.3). We also observed no correlation between JHDM2A mRNA content and the PRM mRNA ratio (R=0.2, P=0.3). Conclusion We found significant correlation between the aberrant PRM ratio (PRM2 under expression) and lower YBX2 mRNA content in testicular biopsies of azoospermic men compared to controls, which suggested that downregulation of the YBX2 gene might be involved in PRM2 under expression. These molecules could be useful biomarkers for predicting male infertility. PMID:26644857

  8. Experiment K-6-11. Actin mRNA and cytochrome c mRNA concentrations in the tricepts brachia muscle of rats

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Morrison, P. R.; Thomason, D. B.; Oganov, V. S.

    1990-01-01

    It is well known that some skeletal muscles atrophy as a result of weightlessness (Steffen and Musacchia 1986) and as a result of hindlimb suspension (Tischler et al., 1985, Thomason et al., 1987). Because the content of protein is determined by the rates of protein synthesis and degradation, a decrease in protein synthesis rate, or an increase in the protein degradation, or changes in both could produce the atrophy. Indeed, an increased protein degradation (Tischler et al., 1985) and a decreased protein synthesis (Thomason et al., 1988) have been observed in skeletal muscles of suspended hindlimbs of rats. Any decrease in protein synthesis rate could be caused by decreases in mRNA concentrations. Such decreases in the concentration and content of alpha-actin mRNA and cytochrome c mRNA have been noted in skeletal muscles of hindlimb suspended rats (Babij and Booth, 1988). From these findings researchers hypothesized that alpha-actin mRNA and cytochrome c mRNA would decrease in the triceps brachia muscle of Cosmos 1887 rats.

  9. Poly(rC) binding proteins mediate poliovirus mRNA stability.

    PubMed Central

    Murray, K E; Roberts, A W; Barton, D J

    2001-01-01

    The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA. PMID:11497431

  10. Cocaine modulates mu-opioid receptor mRNA but not c-fos mRNA levels in primary cortical astrocytes.

    PubMed

    Festa, Eugene D; Cecala, Christine; Quinones-Jenab, Vanya; Jenab, Shirzad

    2002-07-01

    Cocaine is known to modulate the opioid system in several brain regions, including the cortex. Glial cells that are derived from the neonatal cortex have been shown to express opioid peptides and opioid receptors. In this study we investigated the effects of cocaine on c-fos and mu-opioid receptor mRNA levels in primary cortical astrocyte cultures, using RT-PCR and quantitative solution hybridization assays. Astrocyte cultures from 1-day-old Fischer rats were untreated or treated with cocaine for 30min, 2h, or 5h. While c-fos mRNA levels did not change at any time, mu-opioid receptor mRNA levels decreased by 75% after 2 and 5h of cocaine treatments. Our data suggest that cocaine differentially modulates c-fos and opioid signaling in astrocyte cell culture. PMID:12128154

  11. A nucleic acid biosensor for gene expression analysis in nanograms of mRNA.

    PubMed

    Xie, Hong; Yu, Yuan Hong; Xie, Fang; Lao, Yuan Zhi; Gao, Zhiqiang

    2004-07-15

    An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels. PMID:15253638

  12. The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

    PubMed Central

    Haïli, Nawel; Arnal, Nadège; Quadrado, Martine; Amiar, Souad; Tcherkez, Guillaume; Dahan, Jennifer; Briozzo, Pierre; Colas des Francs-Small, Catherine; Vrielynck, Nathalie; Mireau, Hakim

    2013-01-01

    Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria. PMID:23658225

  13. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  14. The DHX33 RNA Helicase Promotes mRNA Translation Initiation

    PubMed Central

    You, Jin; Wang, Xingshun

    2015-01-01

    DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation. PMID:26100019

  15. Transcription control pathways decode patterned synaptic inputs into diverse mRNA expression profiles.

    PubMed

    Jain, Pragati; Bhalla, Upinder S

    2014-01-01

    Synaptic plasticity requires transcription and translation to establish long-term changes that form the basis for long term memory. Diverse stimuli, such as synaptic activity and growth factors, trigger synthesis of mRNA to regulate changes at the synapse. The palette of possible mRNAs is vast, and a key question is how the cell selects which mRNAs to synthesize. To address this molecular decision-making, we have developed a biochemically detailed model of synaptic-activity triggered mRNA synthesis. We find that there are distinct time-courses and amplitudes of different branches of the mRNA regulatory signaling pathways, which carry out pattern-selective combinatorial decoding of stimulus patterns into distinct mRNA subtypes. Distinct, simultaneously arriving input patterns that impinge on the transcriptional control network interact nonlinearly to generate novel mRNA combinations. Our model combines major regulatory pathways and their interactions connecting synaptic input to mRNA synthesis. We parameterized and validated the model by incorporating data from multiple published experiments. The model replicates outcomes of knockout experiments. We suggest that the pattern-selectivity mechanisms analyzed in this model may act in many cell types to confer the capability to decode temporal patterns into combinatorial mRNA expression. PMID:24787753

  16. Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells

    PubMed Central

    Lavoie, Brigitte; Shenoy, Shailesh M.; Blanchard, Jean-Marie; Singer, Robert H.; Bertrand, Edouard

    2016-01-01

    Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm [14]. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the -actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport. PMID:12546792

  17. Mechanism of decay of the cry1Aa mRNA in Bacillus subtilis.

    PubMed Central

    Vázquez-Cruz, C; Olmedo-Alvarez, G

    1997-01-01

    We undertook the study of the decay process of the cry1Aa mRNA of Bacillus thuringiensis expressed in B. subtilis. The cry1Aa transcript is a 3.7-kb mRNA expressed during sporulation whose transcriptional control has previously been studied in both B. subtilis and B. thuringiensis. We found that the cry1Aa mRNA has a half-life of around 9 min and that its decay occurs through endoribonucleolytic cleavages which result in three groups of high-molecular-weight mRNA intermediates ranging in size from 2.7 to 0.5 kb. A comparative study carried out with Escherichia coli showed a similar pattern of degradation intermediates. Primer extension analysis carried out on RNA from B. subtilis revealed that most cleavages occur within two regions located toward the 5' and 3' ends of the mRNA. The most prominent processing site observed for the cry1Aa mRNA isolated from B. subtilis is only two bases away from that occurring on RNA isolated from E. coli. Most cleavage sites occur at seemingly single-stranded RNA segments rich in A and U nucleotides, suggesting that a common and conserved mechanism may process the cry1Aa mRNA. PMID:9335281

  18. Translation by Ribosomes with mRNA Degradation: Exclusion Processes on Aging Tracks

    NASA Astrophysics Data System (ADS)

    Nagar, Apoorva; Valleriani, Angelo; Lipowsky, Reinhard

    2011-12-01

    We investigate the role of degradation of mRNA on protein synthesis using the totally asymmetric simple exclusion process (TASEP) as the underlying model for ribosome dynamics. mRNA degradation has a strong effect on the lifetime distribution of the mRNA, which in turn affects polysome statistics such as the number of ribosomes present on an mRNA strand of a given size. An average over mRNA of all ages is equivalent to an average over possible configurations of the corresponding TASEPboth before steady state and in steady state. To evaluate the relevant quantities for the translation problem, we first study the approach towards steady state of the TASEP, starting with an empty lattice representing an unloaded mRNA. When approaching the high density phase, the system shows two distinct phases with the entry and exit boundaries taking control of the density at their respective ends in the second phase. The approach towards the maximal current phase exhibits the surprising property that the ribosome entry flux can exceed the maximum possible steady state value. In all phases, the averaging over the mRNA age distribution shows a decrease in the average ribosome density profile as a function of distance from the entry boundary. For entry/exit parameters corresponding to the high density phase of TASEP, the average ribosome density profile also has a maximum near the exit end.

  19. AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation

    PubMed Central

    Lee, Kyung-Ha; Kim, Sung-Hoon; Kim, Hyo-Jin; Kim, Wanil; Lee, Hwa-Rim; Jung, Youngseob; Choi, Jung-Hyun; Hong, Ka Young; Jang, Sung Key; Kim, Kyong-Tai

    2014-01-01

    In the present study, we investigated the 3? untranslated region (UTR) of the mouse core clock gene cryptochrome 1 (Cry1) at the post-transcriptional level, particularly its translational regulation. Interestingly, the 3?UTR of Cry1 mRNA decreased its mRNA levels but increased protein amounts. The 3?UTR is widely known to function as a cis-acting element of mRNA degradation. The 3?UTR also provides a binding site for microRNA and mainly suppresses translation of target mRNAs. We found that AU-rich element RNA binding protein 1 (AUF1) directly binds to the Cry1 3?UTR and regulates translation of Cry1 mRNA. AUF1 interacted with eukaryotic translation initiation factor 3 subunit B and also directly associated with ribosomal protein S3 or ribosomal protein S14, resulting in translation of Cry1 mRNA in a 3?UTR-dependent manner. Expression of cytoplasmic AUF1 and binding of AUF1 to the Cry1 3?UTR were parallel to the circadian CRY1 protein profile. Our results suggest that the 3?UTR of Cry1 is important for its rhythmic translation, and AUF1 bound to the 3?UTR facilitates interaction with the 5? end of mRNA by interacting with translation initiation factors and recruiting the 40S ribosomal subunit to initiate translation of Cry1 mRNA. PMID:24423872

  20. Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis.

    PubMed

    Minami, S A; Collis, P S; Young, E E; Weeks, D P

    1981-04-01

    Flagellar excision in Chlamydomonas reinhardii triggers a rapid and extensive induction of tubulin synthesis. Cloned plasmids, pFT beta 1 and pFT beta 2, carrying cDNA inserts complementary to beta-tubulin mRNA, have been prepared and used to demonstrate a direct requirement for tubulin mRNA synthesis during tubulin induction. Increased tubulin mRNA synthesis is detected within 5 min after deflagellation. During the 45 min peak period of tubulin synthesis, tubulin mRNA accumulates to levels 15- to 35-fold higher than those found in control (non-deflagellated) cells. In addition, there appears to be a direct correlation between tubulin mRNA concentrations and the levels of tubulin production during the induction and deinduction cycle that accompanies flagellar regeneration. Amiprophosmethyl (APM), a compound we reported earlier as a selective inhibitor of tubulin synthesis in deflagellated cells, is shown to block the accumulation of tubulin mRNA following flagellar excision and to cause the rapid loss of tubulin mRNA from cells treated at the peak of induction. PMID:7237546

  1. Nonsense mutations in the human. beta. -globin gene affect mRNA metabolism

    SciTech Connect

    Baserga, S.J.; Benz, E.J. Jr. )

    1988-04-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

  2. Does HIV-1 mRNA 5'-untranslated region bear an internal ribosome entry site?

    PubMed

    Smirnova, Victoria V; Terenin, Ilya M; Khutornenko, Anastasia A; Andreev, Dmitri E; Dmitriev, Sergey E; Shatsky, Ivan N

    2016-02-01

    Unspliced human immunodeficiency virus-1 (HIV-1) mRNA is capped and therefore can be translated via conventional scanning mechanism. In addition, its 5' untranslated region (5'UTR) is thought to function as an internal ribosome entry site (IRES) during G2/M-phase of cell cycle or when cap-dependent translation is inhibited. Recently, customary methods of internal initiation demonstrating have been challenged, and consequently existence of certain IRESs of cellular origin has been put under question. Since a precise knowledge of translation initiation mechanism used by HIV may be important for cure development, presence of the IRES in HIV-1 mRNA demands a careful reexamination using contemporary stringent criteria. The key point of our strategy is to compare translation efficiency of bicistronic mRNA bearing HIV-1 unspliced mRNA 5' UTR in the intercistronic position to that of the corresponding capped monocistronic mRNA. This approach allows determination of internal initiation contribution into the overall level of particular mRNA translation. We found that both in cell-free systems and in cultured cells monocistronic mRNA with HIV-1 unspliced mRNA 5'UTR is translated significantly better than bicistronic one. Importantly, it is also true for G2/M-phase stalled cells or for cells under conditions of inhibited cap-dependent translation. Thus, in our hands contribution of internal ribosome entry into the overall level of translation driven by HIV-1 unspliced mRNA 5'UTR is negligible, and 5'-dependent scanning is a primary mechanism of its translation initiation. PMID:26700150

  3. Aberrant Maspin mRNA Expression is Associated with Clinical Outcome in Patients with Pulmonary Adenocarcinoma.

    PubMed

    Lu, Mingjie; Li, Jun; Huang, Zebo; Du, Yiping; Jin, Shidai; Wang, Jian

    2016-01-01

    BACKGROUND The aim of this study was to investigate the expression level of maspin mRNA in pulmonary adenocarcinoma and to clarify its clinical significance in prediction of prognosis. MATERIAL AND METHODS RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks of 30 pairs of pulmonary adenocarcinoma (AC) tissues and adjacent noncancerous tissues (ANT) and in another 81 AC tissues. Expression of maspin mRNA was tested by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and the potential relationship between maspin mRNA expression and clinic pathological features of AC patients was analyzed. RESULTS The expression of maspin mRNA was upregulated in AC samples compared with the ANT (p<0.001). Patients at advanced clinical stage (III) and patients with lymphatic metastasis showed higher maspin mRNA expression level than those in early-stage patients (I and II) (p=0.038) or with non-lymphatic metastasis (p=0.034). The Kaplan-Meier survival curves indicated that disease-free survival (DFS) was significantly worse in high maspin mRNA expression AC patients (p=0.007). Furthermore, multivariate analysis revealed that the expression of maspin mRNA was an independent prognostic marker for AC (p=0.040). CONCLUSIONS Our study reveals that maspin mRNA was significantly up-regulated in tissues of AC patients. Maspin mRNA may be useful as a new marker of prognosis in AC. PMID:26757744

  4. Differential allele-specific accumulation of bovine kappa-casein mRNA throughout lactation.

    PubMed

    Vachon, Dominic; Britten, Michel; Morisset, Jean; Petitclerc, Denis; Robitaille, Gilles

    2004-11-01

    A differential allele-specific accumulation of kappa-casein mRNA that is not linked to the kappa-casein protein variants is described in Holstein cows. Actually, cows genotyped kappa-casein AB were a mixed population. For the first group of kappa-casein AB cows, allele A-specific kappa-casein mRNA contents within mammary epithelial cells were lower than the allele B-specific ones (cows LH), suggesting that the allele A-specific kappa-casein gene was expressed with lower efficiency in mRNA. For the other group of kappa-casein AB cows, allele A- and B-specific kappa-casein mRNA accumulated to a similar level within mammary epithelial cells (cows HH). The objective of this study was to determine whether the accumulation of allele-specific kappa-casein mRNA remained constant throughout lactation for the two groups of cows. Quantitative RT-PCR was used to monitor Holstein cows kappa-casein AB genotyped HH and LH throughout lactation for the proportion of allele B-specific mRNA accumulation relative to the total kappa-casein encoded mRNA within mammary epithelial cells: RNA was extracted from milk somatic cells known to contain a small proportion of mammary epithelial cells. Mean values of allele B-specific mRNA content were 50.6+/-0.5 and 54.0+/-0.9%, for cows HH and cows LH, respectively, and did not vary during lactation (P> 0.10). This suggests that the phenotypic expression of the genetic mutation that causes the differential allele-specific accumulation of kappa-casein mRNA was not affected by physiological and environmental factors, which tend to vary considerably throughout lactation. PMID:15605706

  5. Negative Regulation of Neuromedin U mRNA Expression in the Rat Pars Tuberalis by Melatonin

    PubMed Central

    Aizawa, Sayaka; Sakata, Ichiro; Nagasaka, Mai; Higaki, Yuriko; Sakai, Takafumi

    2013-01-01

    The pars tuberalis (PT) is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD), such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU) that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2) mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm. PMID:23843987

  6. Aberrant Maspin mRNA Expression is Associated with Clinical Outcome in Patients with Pulmonary Adenocarcinoma

    PubMed Central

    Lu, Mingjie; Li, Jun; Huang, Zebo; Du, Yiping; Jin, Shidai; Wang, Jian

    2016-01-01

    Background The aim of this study was to investigate the expression level of maspin mRNA in pulmonary adenocarcinoma and to clarify its clinical significance in prediction of prognosis. Material/Methods RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks of 30 pairs of pulmonary adenocarcinoma (AC) tissues and adjacent noncancerous tissues (ANT) and in another 81 AC tissues. Expression of maspin mRNA was tested by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and the potential relationship between maspin mRNA expression and clinic pathological features of AC patients was analyzed. Results The expression of maspin mRNA was upregulated in AC samples compared with the ANT (p<0.001). Patients at advanced clinical stage (III) and patients with lymphatic metastasis showed higher maspin mRNA expression level than those in early-stage patients (I and II) (p=0.038) or with non-lymphatic metastasis (p=0.034). The Kaplan-Meier survival curves indicated that disease-free survival (DFS) was significantly worse in high maspin mRNA expression AC patients (p=0.007). Furthermore, multivariate analysis revealed that the expression of maspin mRNA was an independent prognostic marker for AC (p=0.040). Conclusions Our study reveals that maspin mRNA was significantly up-regulated in tissues of AC patients. Maspin mRNA may be useful as a new marker of prognosis in AC. PMID:26757744

  7. Message-specific sequestration of maternal histone mRNA in the sea urchin egg.

    PubMed Central

    Showman, R M; Wells, D E; Anstrom, J; Hursh, D A; Raff, R A

    1982-01-01

    Nucleate and anucleate fragments of sea urchin eggs were prepared by centrifugation on sucrose step gradients. The amount of total RNA, poly(A)+ RNA, histone mRNA, actin mRNA, alpha-tubulin mRNA, and mitochondrial rRNA was determined for each fragment. Total RNA, poly(A)+ RNA, actin mRNA, and alpha-tubulin mRNA all distributed in the same ratio as the volume of the fragments. In contrast, the mitochondrial rRNA was found preferentially distributed in the anucleate fragments, coinciding with the distribution of the mitochondria. Histone mRNAs did not follow the fragment volume ratios, but rather were always found associated with the fragment containing the nucleus. To distinguish between nuclear association and possible artifacts associated with centrifugation, eggs were manually cut into nucleate and anucleate fragments and the amount of histone mRNA was determined for each set. Again only the fragments containing the nucleus had detectable amounts of histone mRNA. Although histone mRNAs were always associated with the nucleate fragment, very little histone mRNA was found associated with isolated egg nuclei prepared under gentle isotonic isolation conditions. Furthermore, embryos that have had first nuclear breakdown blocked with 6-dimethylaminopurine still initiated the recruitment of histone mRNAs into polysomes at the same time as control embryos, thus indicating that nuclear breakdown is not necessary for normal histone message utilization. These results demonstrate a message-specific sequestration of maternal histone mRNA which is physically different from that of other maternal mRNAs and which may govern the timing of maternal histone synthesis in sea urchin embryos. Images PMID:6193511

  8. Pros and cons of pDNA and mRNA transfection to study mRNA translation in mammalian cells.

    PubMed

    Andreev, Dmitry E; Terenin, Ilya M; Dmitriev, Sergey E; Shatsky, Ivan N

    2016-03-01

    Protein synthesis in eukaryotes is subject to stringent control. The misregulation of translation of certain mRNAs is often a hallmark of many diseases, including malignancies and autoimmune disorders. To understand why and how it happens, it is important to investigate the translational control of specific mRNAs. In this case, one could use reporter mRNAs in order to identify cis-acting elements responsible for regulation. Here we overview plasmid DNA (pDNA) and mRNA transfections, their pitfalls and limitations, as well as some emerging applications for mRNA transfection. PMID:26680098

  9. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    PubMed

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Mller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention. PMID:26614670

  10. The interleukin 2 receptor (IL-2R): the IL-2R alpha subunit alters the function of the IL-2R beta subunit to enhance IL-2 binding and signaling by mechanisms that do not require binding of IL-2 to IL-2R alpha subunit.

    PubMed

    Grant, A J; Roessler, E; Ju, G; Tsudo, M; Sugamura, K; Waldmann, T A

    1992-03-15

    Interleukin 2 (IL-2)-mediated signaling through its high-affinity receptor involves a complex interrelationship between IL-2 and two IL-2-binding chains, IL-2R alpha and beta chains. Previously with the reagents available it was difficult to define functional interactions between these two IL-2R subunits involved in IL-2 binding and signal transduction. To extend our understanding of the interplay between the two binding subunits we have done studies with the monoclonal antibody HIEI, which interferes with interaction of IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively). Furthermore, we used two forms of IL-2, recombinant native IL-2 and F42A, an IL-2 analog (Phe-42----Ala substitution) that binds only to IL-2R beta. Analog F42A manifested 75-100% of the bioactivity of wild-type IL-2. This observation is inconsistent with the strict hierarchical IL-2-binding affinity conversion model previously proposed by Saito and coworkers [Saito Y., Sabe, H., Suzuki, N., Kondo, S., Ogura, T., Shimizu, A. & Honjo, T. (1988) J. Exp. Med. 168, 1563-1572] that predicted an ordered sequence of events in which IL-2 must first bind to IL-2R alpha before its interaction with IL-2R beta. Previous investigations using IL-2 variants were interpreted to show that IL-2R alpha merely acts to concentrate IL-2 to the cell surface and that no other meaningful interaction occurred between IL-2R alpha and IL-2R beta. However, our data are inconsistent with this view. We draw this conclusion on the basis of our observation that antibody HIEI, which reacts with an epitope of IL-2R alpha and interferes with interaction of this chain and IL-2R beta, inhibits the IL-2-dependent proliferative effects mediated by analog F42A. Furthermore, by blocking interaction of IL-2R alpha and IL-2R beta with the antibody HIEI, a decrease in the affinity of radiolabeled analog F42A for IL-2R beta was seen. In our proposed model IL-2R alpha contributes several functions to IL-2-mediated signaling through the high-affinity IL-2R. These functions include concentration of IL-2 within the two-dimensional surface of the plasma membrane as well as alteration of the functional capacity of IL-2R beta, an effect that does not require prior binding of IL-2R to IL-2R alpha. The IL-2R alpha-mediated augmentation of IL-2R beta functions involves affinity conversion of IL-2R beta, increasing its affinity for IL-2, and may involve facilitation of Il-2-mediated signaling after binding of IL-2 to this IL-2R beta. PMID:1549576

  11. Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA.

    PubMed

    Yanagitani, Kota; Kimata, Yukio; Kadokura, Hiroshi; Kohno, Kenji

    2011-02-01

    Upon endoplasmic reticulum (ER) stress, an endoribonuclease, inositol-requiring enzyme-1?, splices the precursor unspliced form of X-box-binding protein 1 messenger RNA (XBP1u mRNA) on the ER membrane to yield an active transcription factor (XBP1s), leading to the alleviation of the stress. The nascent peptide encoded by XBP1u mRNA drags the mRNA-ribosome-nascent chain (R-RNC) complex to the membrane for efficient cytoplasmic splicing. We found that translation of the XBP1u mRNA was briefly paused to stabilize the R-RNC complex. Mutational analysis of XBP1u revealed an evolutionarily conserved peptide module at the carboxyl terminus that was responsible for the translational pausing and was required for the efficient targeting and splicing of the XBP1u mRNA. Thus, translational pausing may be used for unexpectedly diverse cellular processes in mammalian cells. PMID:21233347

  12. Nerve growth factor mRNA in brain: localization by in situ hybridization

    SciTech Connect

    Rennert, P.D.; Heinrich, G.

    1986-07-31

    Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons.

  13. Screening of Different Organs of Rats for HCA2 Receptor mRNA.

    PubMed

    Shomali, Tahoora; Mosleh, Najmeh; Kamalpour, Mohammad

    2014-01-01

    Interest in hydroxy - carboxylic acid 2 (HCA2) receptor has been raised since it is the target of antidyslipidemic drug nicotinic acid. The present study aimed to evaluate the presence of mRNA of this receptor in different organs of laboratory rat. Twenty two different organs of rats including mesenteric fat, epididymis (head, body and tail), testis, ovary, xiphoid process, liver, adrenal gland, femoral head, proximal epiphyseal and metaphyseal bone marrow of femur, esophagus, glandular stomach, forestomach, intestines, colons, heart, spleen, kidney, trachea, lung, skeletal muscle (quadriceps), cerebrum and cerebellum were removed and examined for HCA2 mRNA by RT- PCR method. The mRNA for HCA2 receptor was detected in all analyzed tissues. In conclusion, the different organs of rat express HCA2 receptor mRNA which makes a proper animal model for future studies on the physiological and pharmacological roles of this receptor in vivo. PMID:25035863

  14. Screening of Different Organs of Rats for HCA2 Receptor mRNA

    PubMed Central

    Shomali, Tahoora; Mosleh, Najmeh; Kamalpour, Mohammad

    2014-01-01

    Interest in hydroxy - carboxylic acid 2 (HCA2) receptor has been raised since it is the target of antidyslipidemic drug nicotinic acid. The present study aimed to evaluate the presence of mRNA of this receptor in different organs of laboratory rat. Twenty two different organs of rats including mesenteric fat, epididymis (head, body and tail), testis, ovary, xiphoid process, liver, adrenal gland, femoral head, proximal epiphyseal and metaphyseal bone marrow of femur, esophagus, glandular stomach, forestomach, intestines, colons, heart, spleen, kidney, trachea, lung, skeletal muscle (quadriceps), cerebrum and cerebellum were removed and examined for HCA2 mRNA by RT- PCR method. The mRNA for HCA2 receptor was detected in all analyzed tissues. In conclusion, the different organs of rat express HCA2 receptor mRNA which makes a proper animal model for future studies on the physiological and pharmacological roles of this receptor in vivo. PMID:25035863

  15. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    NASA Astrophysics Data System (ADS)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  16. Localization of insulin receptor mRNA in rat brain by in situ hybridization.

    PubMed

    Marks, J L; Porte, D; Stahl, W L; Baskin, D G

    1990-12-01

    Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus. PMID:2249648

  17. Free Energy Cost of Stretching mRNA Hairpin Loops Inhibits Small RNA Binding

    PubMed Central

    Meng, Yuzhong; Aalberts, Daniel P.

    2013-01-01

    Small RNA-mRNA binding is an essential step in RNA interference, an important cellular regulatory process. Calculations of binding free energy have been used in binding site prediction, but the cost of stretching the mRNA loop when the small RNA-mRNA duplex forms requires further exploration. Here, using both polymer physics theory and simulations, we estimate the free energy of a stretched mRNA loop. We find loop stretching significantly increases the free energy of 3′ supplementary/compensatory miRNA binding and siRNA binding to mRNA hairpin loops. We also make the observation that sites where 3′ supplementary binding is available may bind at the seed only, and that loop stretching often favors seed-only binding over seed plus 3′ supplementary binding in mRNA hairpins. PMID:23442870

  18. Albumin synthesis in mouse uterus in response to liver mRNA.

    PubMed Central

    Yang, S F; Niu, M C

    1977-01-01

    Messenger RNA was isolated, by means of its attachment to poly(A), from calf liver and the livers of mice, rats, and chickens. When injected into the uterine lumen of immature or spayed mice, both mouse albumin and the albumins characteristic of the species donating the mRNA were synthesized. Studies with inhibitors disclosed that puromycin blocked the synthesis of both albumin species, while dactinomycin affected only mouse albumin synthesis. It appears, therefore, that in the experimental situation used, the function of exogenous liver mRNA is 2-fold: (i) it programs the synthesis of alien albumin in uterine epithelial cells, and (ii) it stimulates the synthesis of mRNA in epithelial cells. The mRNA thus produced primes the synthesis of mouse albumin. Images PMID:325559

  19. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  20. A frameshift mutation prevents Kunitz trypsin inhibitor mRNA accumulation in soybean embryos.

    PubMed Central

    Jofuku, K D; Schipper, R D; Goldberg, R B

    1989-01-01

    We investigated the molecular basis of a soybean Kunitz trypsin inhibitor (KTi) gene mutation that prevents the accumulation of Kunitz trypsin inhibitor protein during seed development. We found that mRNA encoding the major Kunitz trypsin inhibitor protein (KTi3 mRNA) is reduced at least 100-fold in null (KTi-) embryos but that KTi3 gene transcriptional activity is similar in Kunitz trypsin inhibitor producing embryos (KTi+) and in KTi- embryos. We sequenced the Kunitz trypsin inhibitor KTi3 gene from both KTi3+ and KTi3- lines and found that these genes differ by only three nucleotides (+481, +486, and +487) within the KTi3 coding region. Alteration of these nucleotides results in a frameshift within the KTi3- gene that causes premature termination during translation. Our results suggest that the KTi3- frameshift mutation results in KTi3- mRNA destabilization and leads to a drastic reduction in KTi3 mRNA prevalence. PMID:2562563

  1. Luzp4 defines a new mRNA export pathway in cancer cells

    PubMed Central

    Viphakone, Nicolas; Cumberbatch, Marcus G.; Livingstone, Michaela J.; Heath, Paul R.; Dickman, Mark J.; Catto, James W.; Wilson, Stuart A.

    2015-01-01

    Cancer testis antigens (CTAs) represented a poorly characterized group of proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells. Here we show that one CTA, Luzp4, is an mRNA export adaptor. It associates with the TREX mRNA export complex subunit Uap56 and harbours a Uap56 binding motif, conserved in other mRNA export adaptors. Luzp4 binds the principal mRNA export receptor Nxf1, enhances its RNA binding activity and complements Alyref knockdown in vivo. Whilst Luzp4 is up-regulated in a range of tumours, it appears preferentially expressed in melanoma cells where it is required for growth. PMID:25662211

  2. Astrocytes in Alzheimer's disease gray matter express alpha 1-antichymotrypsin mRNA.

    PubMed Central

    Pasternack, J. M.; Abraham, C. R.; Van Dyke, B. J.; Potter, H.; Younkin, S. G.

    1989-01-01

    The serine protease inhibitor alpha 1-antichymotrypsin (ACT) has been shown to be tightly associated with the amyloid found in plaque cores and blood vessels in the brains of patients with Alzheimer's disease (AD). Although the ACT found in plaques could be derived from the high levels of ACT in serum, previous Northern analysis revealed that ACT mRNA is produced locally in AD gray matter at much higher levels than in control gray matter. To determine which brain cells express ACT mRNA, we conducted in situ hybridization with 35S-labeled cRNA probes on hippocampal sections from four AD and three control cases. To identify astrocytes unequivocally, some of the hybridized sections were immunostained for glial fibrillary acidic protein, which is astrocyte-specific. Our results showed numerous astrocytes that were intensely labeled by the probe for ACT mRNA throughout the subicular gray matter of the AD cases. In contrast, astrocytes in control gray matter were rarely labeled by the probe for ACT mRNA. Examination of plaque cores in the AD subiculum showed that some astrocytes intensely labeled by the probe for ACT mRNA were closely associated with virtually every plaque core. Our results also showed many astrocytes in both AD and control white matter that were intensely labeled by the probe for ACT mRNA, and a small fraction of the astrocytes in a juvenile cerebellar astrocytoma that we examined were found to produce high levels of ACT mRNA. In every area in which astrocytes expressing ACT mRNA were found, astrocytes producing no detectable ACT message were also present. Our findings indicate that astrocytes produce the increased ACT mRNA in AD gray matter observed by Northern analysis, but they also show that ACT mRNA expression by astrocytes is not unique to AD. The presence of astrocytes expressing ACT mRNA near, and extending processes towards, plaque cores strongly suggests that some if not all of the ACT associated with amyloid plaque cores is produced by astrocytes surrounding the cores. Images Figure 1 Figure 2 Figure 3 PMID:2817081

  3. Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus.

    PubMed Central

    Zhang, G; Slowinski, V; White, K A

    1999-01-01

    Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3' in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA-RNA interaction. PMID:10199571

  4. A stemloop structure directs oskar mRNA to microtubule minus ends

    PubMed Central

    Jambor, Helena; Mueller, Sandra; Bullock, Simon L.; Ephrussi, Anne

    2014-01-01

    mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stemloop in the oskar 3? UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport. PMID:24572808

  5. Ultra-micro samples can be used for mRNA quantification of lung cancer biomarkers.

    PubMed

    Sandfeld-Paulsen, Birgitte; Demuth, Christina; Folkersen, Birgitte H; Rasmussen, Torben R; Madsen, Line B; Sorensen, Boe S; Meldgaard, Peter

    2016-05-01

    Background Isolating sufficient material for molecular testing remains challenging in non-small cell lung cancer (NSCLC). The use of new ultra-microsamples (uMS) is proven sufficient for DNA and mRNA detection, but whether uMS are useful for quantifying mRNA expression is unknown. We investigated if uMS from lung cancer patients can be used to generate quantitative data on mRNA expression. Methods uMS were collected from primary tumors and lymph nodes from patients suspected of having lung cancer. mRNA was isolated, reverse-transcribed into cDNA and quantified with quantitative PCR assays for hepatocyte growth factor receptor (MET), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR) and amphiregulin (AREG) mRNA. The fraction of tumor cells to normal cells was estimated in each sample. Results MET, HGF, EGFR, and AREG expression were evaluated in 90 samples (30 containing cancer cells and 60 without cancer cells). MET and EGFR expression were negligible in samples without cancer cells. In samples containing cancer cells, MET and EGFR could be quantified in 13 samples each. Adjustment for tumor-cell fraction made it possible to obtain a quantitative result for the tumor-cell mRNA expression of MET and EGFR. In contrast, AREG and HGF were expressed in samples without tumor cells. These samples were used to establish the AREG and HGF mRNA expression in normal cells. Seven out of 14 AR-positive and two out of eight HGF-positive samples with tumor cells were above a cut-off of the mean + 2SD established in samples without tumor cells. Conclusion We demonstrate that uMS contain high-quality mRNA, and quantitative studies can be performed when the tumor-cell fraction is considered. PMID:26923077

  6. TRPM1 (Melastatin-1/MLSN1) mRNA expression in Spitz nevi and nodular melanomas.

    PubMed

    Erickson, Lori A; Letts, Gary A; Shah, Sonali M; Shackelton, Jeffrey B; Duncan, Lyn M

    2009-07-01

    The transient receptor potential cation channel, subfamily M, member 1 (TRPM1/Melastatin-1/MLSN-1) expression has been shown to have prognostic utility in the evaluation of primary cutaneous melanoma. We analyzed a series of spindled and epithelioid cell nevi (Spitz) and primary cutaneous nodular melanomas to determine whether the expression of TRPM1 mRNA may be useful in distinguishing between Spitz nevi and nodular melanomas and to further examine the patterns of TRPM1 mRNA expression in cutaneous melanocytic proliferations. Formalin-fixed, paraffin-embedded tissues from 95 Spitz nevi and 33 nodular melanomas were analyzed for the expression of TRPM1 mRNA by in situ hybridization using (35)S-labeled riboprobes. Ubiquitous melanocytic expression of TRPM1 mRNA was observed in 56 of 95 (59%) Spitz nevi and 4 of 33 (12%) nodular melanomas. Diffusely scattered loss of TRPM1 mRNA was identified in 38 of 95 (40%) Spitz nevi and 2 of 33 (6%) nodular melanomas. Regional loss of the TRPM1 mRNA expression by a significant subset of dermal tumor cells or a complete absence of TRPM1 expression by the dermal tumor was identified in 27 of 33 (82%) nodular melanomas, but only 1 of 95 (1%) Spitz nevi. These findings suggest that the pattern of TRPM1 mRNA expression may be helpful in the differentiation of Spitz nevi and nodular melanomas. Of the 16 patients who experienced metastasis, 15 (94%) had primary tumors that displayed reduced MLSN mRNA expression by all or a part of the dermal tumor. PMID:19396153

  7. Transport and Localization Elements in Myelin Basic Protein mRNA

    PubMed Central

    Ainger, Kevin; Avossa, Daniela; Diana, Amy S.; Barry, Christopher; Barbarese, Elisa; Carson, John H.

    1997-01-01

    Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3′ UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1,473 in the 3′ UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways. PMID:9281585

  8. Effects of ginseng and echinacea on cytokine mRNA expression in rats.

    PubMed

    Ulu???k, Deniz; Keskin, Ercan

    2012-01-01

    The aim of the study was to determine the effect of ginseng and echinacea on the mRNA expression of IL-10, TNF-?, and TGF-?1 in healthy rats. Six-week-old male Fischer 344 rats (n = 48) were used. The animals were divided into three equal groups, as follows: control (C); ginseng (G); echinacea (E). While the C group was fed a standard rat diet (Purina) ad libitum for a period of 40 days, the G and E groups animals received the same diet containing 0.5?g/kg of Panax ginseng root powder and 0.75?g/kg of Echinacea purpurea root powder, respectively. Blood samples were obtained from 8 rats in each group after 20 and 40 days of treatment, and the mRNA expression of IL-10, TNF-?, and TGF-?1 was determined. After 20 days of treatment, the expression of IL-10 mRNA in the G group was different from the C group (P < 0.05); however, after 40 days of treatment, there was no difference between the groups. There was no difference after 20 and 40 days of treatment between the groups with respect to the expression of TGF-?1 mRNA. After 20 days of treatment, the expression of TNF-? mRNA in the E group was higher (P < 0.05) than the C group. After 40 days of treatment, the expression of TNF-? mRNA was similar in all of the groups. Based on the current study, the increase in expression of IL-10 mRNA in the G group and the increase in expression of TNF-? mRNA in the E group support the use of these plants for purposes of modulating the immune system. However, a more detailed study regarding the effects of ginseng and echinacea on these cytokines and other cytokines is needed. PMID:22666172

  9. A stem-loop structure directs oskar mRNA to microtubule minus ends.

    PubMed

    Jambor, Helena; Mueller, Sandra; Bullock, Simon L; Ephrussi, Anne

    2014-04-01

    mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem-loop in the oskar 3' UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport. PMID:24572808

  10. Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient

    PubMed Central

    Zhang, Li; Yang, Zhen; Shi, Bao-Min; Li, Da-Peng; Fang, Chong-Yun; Qiu, Fa-Zu

    2003-01-01

    AIM: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients. METHODS: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis. RESULTS: Expression of local renin mRNA in the liver of control group was (0.19 0.11), significantly lower than that in splenic artery(0.45 0.17)or splenic vein(0.39 0.12) respectively, (P < 0.05). Expression of local angiotensinogen mRNA in the liver was (0.64 0.21), significantly higher than that in splenic artery(0.41 0.15) or in splenic vein (0.35 0.18) respectively, (P < 0.05). Expression of local renin mRNA in the liver, splenic artery and vein of PH group was (0.78 0.28), (0.86 0.35) and (0.81 0.22) respectively, significantly higher than that in the control group, (P < 0.05). Expression of local angiotensinogen mRNA in the liver, splenic artery and vein of PH group was (0.96 0.25), (0.83 0.18) and (0.79 0.23) respectively, significantly higher than that in the control group, (P < 0.05). There was no significant difference between the liver, splenic artery and vein in the expression of local renin or local angiotensinogen mRNA in PH group, (P < 0.05). CONCLUSION: In normal subjects the expression of local renin and angiotensinogen mRNA was organ specific, but with increase of the expression of LRAS, the organ-specificity became lost in cirrhotic patients. LRAS may contribute to increased resistance of portal vein with liver and formation of splanchnic vasculopathy. PMID:12854169

  11. Relating mRNA and protein biomarker levels in a Dehalococcoides and Methanospirillum-containing community.

    PubMed

    Rowe, Annette R; Mansfeldt, Cresten B; Heavner, Gretchen L; Richardson, Ruth E

    2015-03-01

    To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides, an organohalide reducer, and Methanospirillum, a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to theirtemporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167??121 vs. 1095??337 proteins per cell, respectively). In Methanospirillum, MvrD maintained comparable per-cell protein abundance to FrcA (42??14 vs. 60??1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms. PMID:25467924

  12. The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage

    PubMed Central

    Benjamin, Julie-Anna M.; Mass, Eric

    2014-01-01

    Aconitase is an ironsulfur protein and a major enzyme of the TCA cycle that catalyzes the conversion of citrate to isocitrate under iron-rich conditions. In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3?UTR and stabilize it when intracellular iron become scarce. The small regulatory RNA (sRNA) RyhB has previously been shown to promote RNase E-dependent degradation of acnB mRNA when it was expressed from an ectopic arabinose-dependent promoter, independently of intracellular iron levels. In marked contrast, we report here that expression of RyhB under low-iron conditions did not result in acnB mRNA degradation even when RyhB was bound to acnB ribosome binding site (RBS). Genetic and biochemical evidence suggested that, under low-iron conditions, apo-AcnB bound to acnB 3?UTR close to a RNase E cleavage site that is essential for RyhB-induced acnB mRNA degradation. Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site. This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability. PMID:25092924

  13. Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

    PubMed Central

    Gueroussov, Serge; Tarnawsky, Stefan P.; Cui, Xianying A.; Mahadevan, Kohila; Palazzo, Alexander F.

    2010-01-01

    In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data. PMID:21178962

  14. Inhibition of mRNA deadenylation and degradation by ultraviolet light.

    PubMed

    Gowrishankar, Gayatri; Winzen, Reinhard; Bollig, Frank; Ghebremedhin, Beniam; Redich, Natalie; Ritter, Birgit; Resch, Klaus; Kracht, Michael; Holtmann, Helmut

    2005-12-01

    Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light. PMID:16336123

  15. Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation.

    PubMed Central

    Fallon, A M; Jinks, C S; Strycharz, G D; Nomura, M

    1979-01-01

    In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome. Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage. It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon. The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain. Thus, E. coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation. A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism. The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains. PMID:158759

  16. Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

    Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. PMID:25086356

  17. RPFdb: a database for genome wide information of translated mRNA generated from ribosome profiling.

    PubMed

    Xie, Shang-Qian; Nie, Peng; Wang, Yan; Wang, Hongwei; Li, Hongyu; Yang, Zhilong; Liu, Yizhi; Ren, Jian; Xie, Zhi

    2016-01-01

    Translational control is crucial in the regulation of gene expression and deregulation of translation is associated with a wide range of cancers and human diseases. Ribosome profiling is a technique that provides genome wide information of mRNA in translation based on deep sequencing of ribosome protected mRNA fragments (RPF). RPFdb is a comprehensive resource for hosting, analyzing and visualizing RPF data, available at www.rpfdb.org or http://sysbio.sysu.edu.cn/rpfdb/index.html. The current version of database contains 777 samples from 82 studies in 8 species, processed and reanalyzed by a unified pipeline. There are two ways to query the database: by keywords of studies or by genes. The outputs are presented in three levels. (i) Study level: including meta information of studies and reprocessed data for gene expression of translated mRNAs; (ii) Sample level: including global perspective of translated mRNA and a list of the most translated mRNA of each sample from a study; (iii) Gene level: including normalized sequence counts of translated mRNA on different genomic location of a gene from multiple samples and studies. To explore rich information provided by RPF, RPFdb also provides a genome browser to query and visualize context-specific translated mRNA. Overall our database provides a simple way to search, analyze, compare, visualize and download RPF data sets. PMID:26433228

  18. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    SciTech Connect

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-05-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.

  19. Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations.

    PubMed Central

    Primerano, Beatrice; Tassone, Flora; Hagerman, Randi J; Hagerman, Paul; Amaldi, Francesco; Bagni, Claudia

    2002-01-01

    The Fragile X mental retardation gene (FMR1) contains a polymorphic trinucleotide CGG repeat in the 5' untranslated region (UTR) of the FMR1 messenger. We have characterized three lymphoblastoid cell lines derived from unrelated male carriers of a premutation that overexpress FMR1 mRNA and show reduced FMRP level compared to normal cells. The analysis of polysomes/mRNPs distribution of mRNA in the cell lines with a premutation shows that the polysomal association of FMR1 mRNA, which is high in normal cells, becomes progressively lower with increasing CGG repeat expansion. In addition, we could detect a very low level of FMR1 mRNA in a lymphoblastoid cell line from a patient with a full mutation. In this case, FMR1 mRNA is not at all associated with polysomes, in agreement with the complete absence of FMRP. The impairment of FMR1 mRNA translation in patients with the Fragile X syndrome with FMR1 premutation is the cause of the lower FMRP levels that leads to the clinical involvement. PMID:12515381

  20. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    PubMed Central

    Schnell, Steven J.; Ma, Jiong; Yang, Weidong

    2014-01-01

    The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms. PMID:25393401

  1. Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

    PubMed Central

    Ka-Shing-Luk, S.; Kilpatrick, M.; Kerr, K.; Macdonald, P. M.

    1994-01-01

    Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process. PMID:8070663

  2. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    PubMed

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time. PMID:25641752

  3. mRNA transfection of mouse and human neural stem cell cultures.

    PubMed

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Beln Alvarez; Edel, Michael J; Chen, Fred K

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

  4. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

    PubMed Central

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M.; Brandl, Holger; Schwich, Oliver D.; Steiner, Michaela C.; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M.

    2016-01-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends. PMID:26944680

  5. HDAC3 regulates stability of estrogen receptor α mRNA

    SciTech Connect

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn

    2013-03-08

    Highlights: ► HDAC inhibitors decrease the stability of ERα mRNA in MCF-7 cells. ► HDAC3 is involved in maintaining ERα mRNA stability in MCF-7 cells. ► ERα mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ERα-positive MCF-7 cells. ► HDAC3 specific inhibitor will be one of new drugs for ERα-positive breast cancers. -- Abstract: Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

  6. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  7. Differential expression of thyrotropin receptor mRNA in the porcine heart.

    PubMed

    Sellitti, D F; Hill, R; Doi, S Q; Akamizu, T; Czaja, J; Tao, S; Koshiyama, H

    1997-08-01

    Thyrotropin receptor (TSHR) mRNA expression has previously been detected in human heart, suggesting a possible role for the receptor in cardiac function and pathophysiology. In the present study we examined the regional distribution of TSHR mRNA in pig heart to map potential cardiac sites of TSH action. Polyadenylated mRNA extracted from thyroid, atria, ventricles, aorta, coronary arteries, epicardial fat, and purified preparations of atrial and ventricular cardiomyocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) using primers designed to amplify a 311 base pair (bp) DNA segment of the human TSHR. After reverse transcription of 100 ng mRNA, cDNA was amplified by PCR using TSHR primers and compared by electrophoresis on 2% agarose gels. Relative levels of TSHR cDNA (normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) were as follows: Coronary arteries, epicardial fat > right atrium > left atrium > right ventricle, aorta > left ventricle, ventricular cardiocytes. In contrast to ventricular cardiocytes, purified atrial cardiocytes expressed levels of TSHR mRNA readily detectable with RT-PCR. These findings demonstrate that TSHR mRNA expression in porcine heart varies regionally, and furthermore suggest that areas of highest expression (coronary arteries, adipose tissue, right atrium) are potential sites for a functional or pathologic role of the TSHR. PMID:9292956

  8. Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

    PubMed Central

    Spangler, Jacob B.; Feltus, Frank Alex

    2013-01-01

    Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near ? duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near ? duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

  9. Dynactin suppresses the retrograde movement of apically localized mRNA in Drosophila blastoderm embryos

    PubMed Central

    Vendra, Georgia; Hamilton, Russell S.; Davis, Ilan

    2007-01-01

    Motor dependent transport of mRNA is a key mechanism in axis specification during development. Apical transport and anchoring of wingless and pair-rule transcripts in the Drosophila syncytial blastoderm embryo is mediated by cytoplasmic Dynein, the major minus end directed microtubule dependent molecular motor. Here, we show that, despite apical transport of mRNA being highly directional, mRNA particles often pause and move backward toward the plus ends of microtubules. We suggest that this retrograde movement helps overcome cellular obstructions. We show that the plus end movement of apical mRNA is independent of the major plus end microtubule motors Kinesin-1 and Kinesin-2. In contrast, Dynactin, a Dynein processivity factor, is required to suppress retrograde mRNA movements, as well as for efficient minus end motility. We propose that Dynein itself, rather than the activity of a plus end motor, is responsible for the plus end movements of the mRNA and that Dynactin is involved in preventing short reverse movements of the Dynein motor, known to occur in vitro. PMID:17901156

  10. Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

    PubMed

    Saha, Chaitrali; Hegde, Pushpa; Friboulet, Alain; Bayry, Jagadeesh; Kaveri, Srinivas V

    2015-01-01

    Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA. PMID:25664986

  11. mRNA transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers.

    PubMed

    Rejman, Joanna; Tavernier, Geertrui; Bavarsad, Neda; Demeester, Joseph; De Smedt, Stefaan C

    2010-11-01

    Messenger RNA encoding luciferase (mLUC) was complexed to the cationic lipids Lipofectamine or DOTAP/DOPE, and to the cationic polymer linear poly(ethyleneimine) (linPEI). The complexes were incubated with HeLa cells and luciferase expression was assessed. The type of non-viral carrier used determined the extent and duration of protein expression. Maximal duration of mRNA expression was about 9 days for Lipofectamine complexes, i.e. not very much shorter than with pDNA polyplexes. Interestingly, luciferase activity was already detected 30 min after adding the mRNA complexes to the cells, independent on the type of carrier. We also assessed the proportion of cells that become transfected by means of transfection with an mRNA encoding GFP. For both cationic lipids transfection with mRNA yielded a substantially larger fraction of transfected cells (more than 80%) than transfection with pDNA (40%). In addition we tested the carriers for their ability to mediate delivery of mRNA encoding CXCR4 into mesenchymal stem cells. The fraction of CXCR4-positive cells obtained with the mRNA-cationic lipid complexes was around 80%, as compared to 40% for the linPEI polyplexes. Our results demonstrate that the advantage of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period. PMID:20708647

  12. Splicing promotes the nuclear export of ?-globin mRNA by overcoming nuclear retention elements

    PubMed Central

    Akef, Abdalla; Lee, Eliza S.; Palazzo, Alexander F.

    2015-01-01

    Most current models of mRNA nuclear export in vertebrate cells assume that an mRNA must have specialized signals in order to be exported from the nucleus. Under such a scenario, mRNAs that lack these specialized signals would be shunted into a default pathway where they are retained in the nucleus and eventually degraded. These ideas were based on the selective use of model mRNA reporters. For example, it has been shown that splicing promotes the nuclear export of certain model mRNAs, such as human ?-globin, and that in the absence of splicing, the cDNA-derived mRNA is retained in the nucleus and degraded. Here we provide evidence that ?-globin mRNA contains an element that actively retains it in the nucleus and degrades it. Interestingly, this nuclear retention activity can be overcome by increasing the length of the mRNA or by splicing. Our results suggest that contrary to many current models, the default pathway for most intronless RNAs is to be exported from the nucleus, unless the RNA contains elements that actively promote its nuclear retention. PMID:26362019

  13. The human cytomegalovirus regulatory protein UL69 and its effect on mRNA export.

    PubMed

    Toth, Zsolt; Stamminger, Thomas

    2008-01-01

    One of the characteristic features of herpesviruses is that most of their genes are intronless. Thus, their replication relies on the selective nuclear export of intronless viral mRNAs, which have to compete with the nuclear export of bulk spliced cellular mRNAs. Therefore, the regulation of nuclear mRNA export is crucial for the replication and pathogenesis of herpesviruses. Besides the thymidine kinase transcript of herpes simplex virus type 1, which contains specific sequences to facilitate the nuclear export of intronless mRNA, other cis-acting RNA elements for nuclear mRNA export have not yet been identified in the rest of herpesviral intronless mRNAs. Instead, emerging studies show that herpesviruses encode viral mRNA export factors, which interact with components of the major cellular mRNA export pathway, the RNA polymerase II transcription complex and specific splicing factors to selectively export intronless herpesviral mRNAs to the cytoplasm in infected cells. These herpesviral mRNA export factors are members of a conserved gene family that can be found in all herpesviruses. The human cytomegalovirus transactivator protein UL69, which has been demonstrated to belong to this conserved protein family, shares common features with its herpesviral homologues but also possesses unique properties that will be discussed in this review. PMID:17981767

  14. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    PubMed

    Mller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  15. Copper deficiency lowers rat liver glutathione peroxidase activity and mRNA but not selenium

    SciTech Connect

    Prohaska, J.R.; Zinn, K.; Sunde, R.A. )

    1991-03-15

    Copper (Cu) deficiency in rodents leads to lower activity of the liver selenoenzyme glutathione peroxidase (GSH-Px) by an unknown mechanism. Dietary selenium (Se) deficiency is known to be accompanied by proportional loss of GSH-Px activity, protein, and mRNA levels. Lower GSH-Px activity in Cu-deficient ({minus}Cu) rodents might be due to changes in liver Se which could then influence the level of GSH-Px mRNA. Cu deficiency was induced by feeding a diet low in Cu but adequate in Se to Sprague Dawley dams beginning the day of parturition. All {minus}Cu rats had significantly lower liver Cu and cuproenzyme activities than +Cu rats. Portions of liver previously frozen in liquid nitrogen from 60-d-old rats were used to determine Se by neutron activation and for measurement of GSH-Px mRNA. Total RNA was isolated and fractionated by agarose gel electrophoresis and subjected to Northern blot hybridization. Autoradiograms were quantified by densitometry. Compared to +Cu rats the {minus}Cu male and female rats had lower GSH-Px activity and mRNA levels; however, total Se levels were not different. The correlation between GSH-Px activity and mRNA levels was excellent; over a 3-fold range r{sup 2}=0.94. Lower GSH-Px activity and mRNA levels in Cu deficient rats appears not to be due to a direct effect of lower Se levels.

  16. RPFdb: a database for genome wide information of translated mRNA generated from ribosome profiling

    PubMed Central

    Xie, Shang-Qian; Nie, Peng; Wang, Yan; Wang, Hongwei; Li, Hongyu; Yang, Zhilong; Liu, Yizhi; Ren, Jian; Xie, Zhi

    2016-01-01

    Translational control is crucial in the regulation of gene expression and deregulation of translation is associated with a wide range of cancers and human diseases. Ribosome profiling is a technique that provides genome wide information of mRNA in translation based on deep sequencing of ribosome protected mRNA fragments (RPF). RPFdb is a comprehensive resource for hosting, analyzing and visualizing RPF data, available at www.rpfdb.org or http://sysbio.sysu.edu.cn/rpfdb/index.html. The current version of database contains 777 samples from 82 studies in 8 species, processed and reanalyzed by a unified pipeline. There are two ways to query the database: by keywords of studies or by genes. The outputs are presented in three levels. (i) Study level: including meta information of studies and reprocessed data for gene expression of translated mRNAs; (ii) Sample level: including global perspective of translated mRNA and a list of the most translated mRNA of each sample from a study; (iii) Gene level: including normalized sequence counts of translated mRNA on different genomic location of a gene from multiple samples and studies. To explore rich information provided by RPF, RPFdb also provides a genome browser to query and visualize context-specific translated mRNA. Overall our database provides a simple way to search, analyze, compare, visualize and download RPF data sets. PMID:26433228

  17. Viscum album-Mediated COX-2 Inhibition Implicates Destabilization of COX-2 mRNA

    PubMed Central

    Saha, Chaitrali; Hegde, Pushpa; Friboulet, Alain; Bayry, Jagadeesh; Kaveri, Srinivas V.

    2015-01-01

    Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1?-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA. PMID:25664986

  18. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    SciTech Connect

    Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 ; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 ; Chand, Ashwini L.; Clyne, Colin D.

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  19. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    PubMed

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. PMID:26410240

  20. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development

    PubMed Central

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance. PMID:26379701

  1. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  2. Prognostic value of ISG15 mRNA level in drinkers with esophageal squamous cell cancers

    PubMed Central

    Tao, Jun; Hua, Ping; Wen, Jing; Hu, Yi; Yang, Hong; Xie, Xuan

    2015-01-01

    ISG15, the protein encoded by interferon (IFN)-stimulated gene 15, was the first identified ubiquitin-like protein, which could be strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. Although the biological activities of ISG15 have yet to be fully elucidated, it is frequently overexpressed in various cancers. As the role of ISG15 in esophageal squamous cell cancer (ESCC) has not been well reported, the current study aimed to elucidate the role of ISG15 in predicting outcomes of ESCC patients. Samples were collected from 153 ESCC patients, including 54 pairs of tumor tissues and non-tumor tissues. Compared with the paired non-tumor tissues, higher expression of ISG15 mRNA were detected in ESCC tissues. The cut-off value 1.28 determined by ROC curve analysis divided the ESCC patients into high and low ISG15 mRNA expression group. High-ISG15 mRNA expression appeared with more frequency in ever-drinkers (P = 0.018). Kaplan-Meier analysis indicated that Low-ISG15 mRNA expression group had a longer cancer-specific survival (CSS) compared with High-ISG15 mRNA expression group. Multivariate analysis revealed that ISG15 mRNA (P = 0.024; hazard ratio, 2.759, 95% CI, 1.841-4.134) as well as Pathological staging (P < 0.001; hazard ratio, 1.634, 95% CI, 1.065-2.505) were independent prognostic factors. Subgroup analysis revealed that the discernibility of ISG15 mRNA level on ESCC outcomes was only pronounced in ever-drinkers (P = 0.026) not in never-drinkers (P = 0.138). ISG15 might serve as a novel prognostic biomarker in drinkers with ESCC. PMID:26617815

  3. Mapping of N6-methyladenosine residues in bovine prolactin mRNA.

    PubMed Central

    Horowitz, S; Horowitz, A; Nilsen, T W; Munns, T W; Rottman, F M

    1984-01-01

    N6-Methyladenosine (m6A) residues, which are found internally in viral and cellular mRNA populations at the sequences Apm6ApC and Gpm6ApC, have been proposed to play a role in mRNA processing and transport. We have developed a sensitive approach to analyze the level and location of m6A in specific purified cellular mRNAs in an attempt to correlate m6A location with function. Polyadenylylated mRNA is hybridized to cDNA clones representing the full size mRNA under study or fragments of it, and the protected RNA is digested and labeled with polynucleotide kinase in vitro. After enrichment for m6A with anti-m6A antibody, the [32P]-pm6A is separated on TLC plates, and compared with the total amount of radiolabeled nucleotides. Using this combination of in vitro RNA labeling and antibody selection, we were able to detect m6A in purified stable mRNAs that cannot be readily labeled in cells with greater sensitivity than was possible by previous techniques. We applied this technique to bovine prolactin mRNA and showed that this mRNA contains m6A. Moreover, all of the m6A residues in this message are found within the 3' two-thirds of the molecule and are highly concentrated (61%) within a sequence of 108 nucleotides at the 3' noncoding region of the message. The nonrandom distribution of m6A in a specific cellular mRNA, as demonstrated for bovine prolactin, will have to be taken into account when designing a model for m6A function. Images PMID:6592581

  4. High performance mRNA transfection through carbonate apatite-cationic liposome conjugates.

    PubMed

    Zohra, Fatema T; Chowdhury, Ezharul H; Akaike, Toshihiro

    2009-08-01

    mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Delivery of mRNA can bypass nuclear localization step enabling protein expression directly in cytoplasm through transcription. Current technologies for mRNA delivery are predominantly based on cationic liposomes with low activity for transfection. We, previously reported that applying inorganic nano-particles of carbonate apatite onto cationic liposome of DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} resulted in high transfection potency for luciferase mRNA both in mitotic and non-mitotic cells. In this paper, we expanded the previous work and performed in detail study especially on two important parts, evaluating the image of the complex and analyzing the steps of gene delivery to detect the determinant factor for enhanced transfection potency. Transmission electron microscopic (TEM) observation clearly indicated the presence of inorganic carbonate apatite particles on mRNA-liposome complex and demonstrated the structure of the new hybrid carrier material. Due to apparently higher gravitational force of absorbed inorganic nano-particles, cellular contact and internalization of hybrid-particle-associated mRNA were significantly enhanced compared to DOTAP. This analysis indicates rather than downstream steps, initial steps of cell membrane binding and subsequent way of internalization could be the determinant factor for final protein expression. Moreover, we compared transfection efficiency of mRNA and pDNA in Human Umbilical Vein Endothelial cell (HUVEC) to demonstrate advantages of mRNA delivery. PMID:19410288

  5. Lipoprotein lipase and hepatic lipase mRNA tissue specific expression, developmental regulation, and evolution.

    PubMed

    Semenkovich, C F; Chen, S H; Wims, M; Luo, C C; Li, W H; Chan, L

    1989-03-01

    Lipoprotein lipase (LPL) and hepatic lipase (HL) enzyme activities were previously reported to be regulated during development, but the underlying molecular events are unknown. In addition, little is known about LPL evolution. We cloned and sequenced a complete mouse LPL cDNA. Comparison of sequences from mouse, human, bovine, and guinea pig cDNAs indicated that the rates of evolution of mouse, human, and bovine LPL are quite low, but guinea pig LPL has evolved several times faster than the others. 32P-Labeled mouse LPL and rat HL cDNAs were used to study lipase mRNA tissue distribution and developmental regulation in the rat. Northern gel analysis revealed the presence of a single 1.87 kb HL mRNA species in liver, but not in other tissues including adrenal and ovary. A single 4.0 kb LPL mRNA species was detected in epididymal fat, heart, psoas muscle, lactating mammary gland, adrenal, lung, and ovary, but not in adult kidney, liver, intestine, or brain. Quantitative slot-blot hybridization analysis demonstrated the following relative amounts of LPL mRNA in rat tissues: adipose, 100%; heart, 94%; adrenal, 6.6%; muscle, 3.8%; lung, 3.0%; kidney, 0%; adult liver, 0%. The same quantitative analysis was used to study lipase mRNA levels during development. There was little postnatal variation in LPL mRNA in adipose tissue; maximal levels were detected at the earliest time points studied for both inguinal and epididymal fat. In heart, however, LPL mRNA was detected at low levels 6 days before birth and increased 278-fold as the animals grew to adulthood.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2723548

  6. Circulating resistin protein and mRNA concentrations and clinical severity of coronary artery disease

    PubMed Central

    Sopic, Miron; Spasojevic-Kalimanovska, Vesna; Kalimanovska-Ostric, Dimitra; Andjelkovic, Kristina; Jelic-Ivanovic, Zorana

    2015-01-01

    Introduction Previous studies have implicated a strong link between circulating plasma resistin and coronary artery disease (CAD). The aim of this study was to evaluate the differences in peripheral blood mononuclear cells (PBMC) resistin mRNA and its plasma protein concentrations between the patients with CAD of different clinical severity. Material and methods This study included 33 healthy subjects as the control group (CG) and 77 patients requiring coronary angiography. Of the latter 30 was CAD negative whereas 47 were CAD positive [18 with stable angina pectoris (SAP) and 29 with acute coronary syndrome (ACS)]. Circulating resistin was measured by ELISA; PBMC resistin mRNA was determined by real-time PCR. Results Resistin protein was significantly higher in the ACS group compared to the CG (P = 0.001) and the CAD negative group (P = 0.018). Resistin mRNA expression did not vary across the study groups, despite the positive correlation seen with plasma resistin (? = 0.305, P = 0.008). In patients, plasma resistin and PBMC resistin mRNA negatively correlated with HDL-C (? = -0.404, P < 0.001 and ? = -0.257, P = 0.032, respectively). Furthermore, the highest plasma resistin tertile showed the lowest HDL-C (P = 0.006). Plasma resistin was positively associated with serum creatinine (? = 0.353, P = 0.002). Conclusion Significant increase of plasma resistin in patients with ACS compared to CG and CAD negative patients was observed. Despite no change in PBMC resistin mRNA in different disease conditions a positive association between resistin mRNA and resistin plasma protein was evident. Both plasma resistin and PBMC resistin mRNA were negatively associated with plasma HDL-C, and plasma resistin positively with serum creatinine. PMID:26110037

  7. Urinary vimentin mRNA as a potential novel biomarker of renal fibrosis.

    PubMed

    Cao, Yu-Han; Lv, Lin-Li; Zhang, Xu; Hu, Hong; Ding, Li-Hong; Yin, Di; Zhang, Ying-Zi; Ni, Hai-Feng; Chen, Ping-Sheng; Liu, Bi-Cheng

    2015-09-15

    Renal fibrosis is a histological outcome of chronic kidney disease (CKD) progression. However, the noninvasive detection of renal fibrosis remains a challenge. Here we constructed a renal fibrosis target mRNA array and used it to detect urinary mRNAs of CKD patients for investigating potential noninvasive biomarkers of renal fibrosis. We collected urine samples from 39 biopsy-proven CKD patients and 11 healthy controls in the training set. Urinary mRNA profiles of 86 genes showed a total of 21 mRNAs that were differentially expressed between CKD patients and controls (P < 0.05), and vimentin (VIM) mRNA demonstrated the highest change fold of 9.99 in CKD vs. controls with robust correlations with decline of renal function and severity of tubulointerstitial fibrosis. Additionally, VIM mRNA further differentiated patients with moderate-to-severe fibrosis from none-to-mild fibrosis group with an area of the curve of 0.796 (P = 0.008). A verification of VIM mRNA in the urine of an additional 96 patients and 20 controls showed that VIM is not only well correlated with renal function parameters but also correlated with proteinuria and renal fibrosis scores. Multiple logistic regression and receiver-operating characteristics analysis further showed that urine VIM mRNA is the best predictive parameter of renal fibrosis compared with estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen. In addition, there is no improved predictive performance for the composite biomarkers to predict renal fibrosis severity compared with a single gene of VIM. Overall, urinary VIM mRNA might serve as a novel independent noninvasive biomarker to monitor the progression of kidney fibrosis. PMID:25904701

  8. A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

    PubMed

    Muller, Mandy; Hutin, Stephanie; Marigold, Oliver; Li, Kathy H; Burlingame, Al; Glaunsinger, Britt A

    2015-05-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA. PMID:25965334

  9. A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases

    PubMed Central

    Muller, Mandy; Hutin, Stephanie; Marigold, Oliver; Li, Kathy H.; Burlingame, Al; Glaunsinger, Britt A.

    2015-01-01

    During lytic Kaposis sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3 untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3 UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3 UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA. PMID:25965334

  10. Molecular cloning and regulation of murine fatty acid synthase mRNA

    SciTech Connect

    Paulauskis, J.D.; Sul, H.S.

    1987-05-01

    Mouse liver mRNA that was enriched in sequences coding for fatty acid synthase (FAS) by sucrose-density gradient centrifugation was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into pBR322 and lambdagt10 and cloned. Clones containing putative cDNA sequences for FAS were identified by differential hybridization where /sup 32/P-cDNAs, synthesized from sucrose gradient purified liver mRNA from mice starved or starved and refed a fat-free diet, were used as probes. Two of these clones were further studied and found to contain sequences complementary to FAS mRNA by hybrid-selected translation and specific immunoprecipitation. Using these clones as probes, they selected 33 additional clones containing cDNA sequences for FAS. Partial DNA sequence data for these clones were obtained. Northern blot analysis revealed a single mRNA size of 9.3 kb when a cDNA clone with a 3.1 kb insert was used as a probe. This is in contrast to rat liver FAS which showed two mRNAs sizes of 9.2 and 10.0 kb. They also studied FAS mRNA level of 3T3-L1 preadipocytes during differentiation into adipocytes. An approximate 10-fold increase in FAS mRNA content was observed which corresponded with an increased rate of FAS synthesis indicating pretranslational regulation. The FAS cDNA probe was also employed to demonstrate that induction of FAS in the livers of previously starved mice that were fed a fat-free diet was controlled pretranslationally by a parallel modulation of the FAS mRNA concentration.

  11. Monitoring Therapeutic Efficacy by Real-Time Mycobacterium tuberculosis mRNA Detection in Sputum

    PubMed Central

    Mdivani, Nino; Li, Haijing; Akhalaia, Maka; Gegia, Medea; Goginashvili, Leila; Kernodle, Douglas S.; Khechinashvili, George; Tang, Yi-Wei

    2010-01-01

    BACKGROUND Current laboratory methods for monitoring response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify response to anti-TB drug efficacy are desirable. METHODS We developed two real-time PCR assays using hydrolysis probes to target DNA of the IS6110 insertion element and mRNA of antigen 85B, respectively, extracted directly from sodium hydroxide-N-acetyl-L-cysteine decontaminated and concentrated sputum specimens. We prospectively compared these assays to sputum mycobacterial culture in patients receiving anti-TB therapy. RESULTS Sixty-five patients with newly diagnosed tuberculosis and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2, and months 1, 2 and 4 after initiating therapy. Both DNA PCR (98.5% positive) and mRNA RT-PCR (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum specimens. Overall agreement between culture and mRNA RT-PCR among all 286 sputum specimens was 87.1%, and compared to culture the mRNA RT-PCR diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled culture results at the follow-up time points. CONCLUSIONS The continued presence of viable M. tuberculosis by culture and antigen 85B mRNA by RT-PCR correlated clinically with anti-TB drug resistance, whereas the DNA PCR assay showed a high false positive rate. This mRNA RT-PCR assay may allow rapid monitoring of response to anti-TB therapy. PMID:19574468

  12. Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia

    PubMed Central

    Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua

    2014-01-01

    Introduction To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Material and methods Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). Results The average H19 mRNA level of the macrosomia group was 1.450 0.456 while in the control group it was 2.080 1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.020.59) when compared to those in the lowest tertile (p for linear trend = 0.009). Conclusions The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia. PMID:25097584

  13. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    SciTech Connect

    Dalgaard, Louise T.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  14. Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

    PubMed Central

    2015-01-01

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  15. Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

    PubMed Central

    Jakobson, M; Jakobson, M; Llano, O; Palgi, J; Arumäe, U

    2013-01-01

    N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5′-untranslated region (UTR) and naturally spliced exon–exon junction downstream of the premature translation termination codon in the 3′UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response. PMID:23969856

  16. A transgenic mouse for in vivo detection of endogenous labeled mRNA

    PubMed Central

    Lionnet, Timothée; Czaplinski, Kevin; Darzacq, Xavier; Shav-Tal, Yaron; Wells, Amber L.; Chao, Jeffrey A.; Park, Hye Yoon; de Turris, Valeria; Lopez-Jones, Melissa; Singer, Robert H.

    2010-01-01

    Live-cell single mRNA imaging is a powerful tool, but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line in which an MS2 binding site (MBS) cassette was targeted to the 3′UTR of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we were able to uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real-time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We performed tracking of single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS also provided a means for high sensitivity Fluorescence In Situ Hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues. PMID:21240280

  17. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus

    SciTech Connect

    Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.

    1987-10-01

    Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

  18. Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review).

    PubMed

    Di Liegro, Carlo Maria; Schiera, Gabriella; Di Liegro, Italia

    2014-04-01

    Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5' or 3' to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3'-untranslated (3'-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs. PMID:24452120

  19. Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system.

    PubMed

    Nosrat, C A; Tomac, A; Lindqvist, E; Lindskog, S; Humpel, C; Strmberg, I; Ebendal, T; Hoffer, B J; Olson, L

    1996-11-01

    Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-beta family and has potent neurotrophic effects on several classes of neurons including dopamine neurons and motoneurons. Here, we have used in situ hybridization to describe the development of the cellular expression of GDNF mRNA pre- and postnatally. Consistent with dopaminotrophic activity, GDNF mRNA is expressed in the developing basal ganglia and the olfactory tubercle. It is also found in a thalamic nucleus, in neurons of the substantia innominata, in the developing Purkinje neurons and the developing locus coeruleus area, and in trigeminal brainstem nuclei. In the spinal cord, neuronal expression is found in Clarke's column. GDNF mRNA is also expressed in the dorsal horns during development. Additional GDNF mRNA expression in the head region includes the carotid body, the retina, the vibrissae, the inner ear, the ear canal, and epitheli