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Sample records for jnk-mediated interleukin-2 mrna

  1. Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages.

    PubMed

    Varì, Rosaria; D'Archivio, Massimo; Filesi, Carmelina; Carotenuto, Simona; Scazzocchio, Beatrice; Santangelo, Carmela; Giovannini, Claudio; Masella, Roberta

    2011-05-01

    Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences. PMID:20621462

  2. 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

  3. 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

  4. 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

  5. Genistein upregulates LDLR levels via JNK-mediated activation of SREBP-2

    PubMed Central

    Kartawijaya, Medicia; Han, Hye Won; Kim, Yunhye; Lee, Seung-Min

    2016-01-01

    Background Genistein has been proved in vitro and in vivo to lower LDLR level. It is also widely consumed and implicated for its anti-atherogenic effects. However, the molecular mechanism by which genistein lowers the LDL level is still unknown. Objective To understand the anti-atherogenic molecular mechanism of action, genistein was investigated for its impact on the expression of LDLR, the receptor for LDL cholesterol, and related signaling pathways in a human hepatoma cell line. Design HepG2 cell was used for the experiments. Genistein with different concentrations was diluted in media and was incubated for 24 h or more as indicated. Protein levels were measured by western blotting, and mRNA expression was detected by RT-qPCR. Chromatin immunoprecipitation assay (CHIP) assay was used to determine protein binding levels, and luciferase assay was used to measure promoter activity. Result Genistein increased the mRNA and protein levels of LDLR in a time-dependent manner. Genistein increased the transcriptional activity of the LDLR promoter containing the reporter gene (pLDLR-luc, −805 to +50). But the sterol regulatory element deletion mutant construct failed to be activated by genistein. Genistein increased the nuclear fraction of SREBP-2 and the DNA-binding activity of SREBP-2 to LDLR promoter, as assessed by CHIP. The genistein-phosphorylated JNK inhibitor (SP600126) abolished the genistein-stimulated levels of LDLR and the nuclear SREBP-2. The addition of cholesterol up to 5 µg/mL for 24 h did not affect the effect of genistein on LDLR protein expression. Even the addition of 40 µM genistein increased the cholesterol uptake by more than 10% in the human hepatoma cell line. Conclusion Our data support the idea that genistein may have anti-atherogenic effects by activating JNK signals and SREBP-2 processing, which is followed by the upregulation of LDLR. PMID:27211318

  6. 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

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

    PubMed Central

    Ngoc, Tam Dan Nguyen; Son, Young-Ok; Lim, Shin-Saeng; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

    2012-01-01

    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 G2/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. PMID:22285274

  8. 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; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

    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.

  9. Enhancement of interleukin-2 activity by liposomes.

    PubMed

    Joffret, M L; Morgeaux, S; Laclerc, C; Oth, D; Zanetti, C; Sureau, P; Perrin, P

    1990-08-01

    The present report demonstrates that liposomes increase the interleukin-2 (IL-2) dependent proliferation of cytotoxic T-lymphocyte line (CTLL) cells used for the measurement of IL-2 activity. This effect was better observed with suboptimal doses of IL-2 and low concentrations of lipids. The increased IL-2 dependent proliferation is not due to a direct effect of liposomes on CTLL cells but rather to an interaction between IL-2 and liposomes. An interaction between IL-2 and components of fetal calf serum is also demonstrated. The results indicate that liposomes may interfere with IL-2 bioassay but also show the possibility of potentiating IL-2 activity for therapeutic purposes. PMID:2396476

  10. 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

  11. Impaired production of interleukin-2 after surgery.

    PubMed Central

    Akiyoshi, T; Koba, F; Arinaga, S; Miyazaki, S; Wada, T; Tsuji, H

    1985-01-01

    The capacity of peripheral blood mononuclear cells (PBM) to produce interleukin-2 (IL-2) was studied serially before and following operation in patients undergoing various surgical procedures. In patients who had major surgery, significant decrease in IL-2 activity was observed 1, 3 and 6 days after operation as compared to that before surgery, although there was no significant change throughout the post-operative course in patients undergoing minor surgery. IL-2 activity returned to the pre-operative level by the 8th post-operative day. However, it remained significantly depressed 8 days after surgery in patients who had undergone major surgical procedures of more increasing severity. Distribution of T cell subsets, especially OKT4 positive cells, did not differ significantly from the pre-operative value throughout the post-operative course. However, the depressed production of IL-2 3 days after surgery could be abolished when adherent cells were removed from PBM by plastic adherence procedures. These results indicated that adherent cells, but not quantitative change in T cell subsets, might be responsible for the depression of IL-2 production after surgery. PMID:3871677

  12. Production of interleukin-2 and interleukin-2 inhibitor in patients with palmoplantar pustulosis.

    PubMed

    Shiohara, T; Kobayashi, M; Ishii, Y; Nagashima, M

    1987-01-01

    We studied production of interleukin-2 (IL-2) and IL-2 inhibitor from peripheral blood of patients with palmoplantar pustulosis (PPP). Nineteen patients were divided into two groups: those with and those without arthro-osteitis. Although IL-2 production in both groups of patients was within normal limits, those with arthro-osteitis showed greater fluctuation in relation to the disease activity. The IL-2 production of five PPP patients with arthro-osteitis was greatly enhanced in the inactive stage compared with the active stage. Sera from two patients treated with a combination of etretinate and colchicine contained extremely low levels of IL-2 inhibitory activity. The increased IL-2 production in the inactive stage may be due in part to the depletion of IL-2 inhibitor-producing cells by the treatment. PMID:2448994

  13. Soluble interleukin-2 receptor α and interleukin-2 serum levels in patients with basal cell carcinoma

    PubMed Central

    Bien, Ewa; Zablotna, Monika; Sokolowska-Wojdylo, Malgorzata; Sikorska, Monika; Lange, Magdalena; Nowicki, Roman

    2016-01-01

    Introduction Basal cell carcinoma (BCC) is an immunogenic neoplasm and the imbalance in Th1/Th2 cytokines expression seems to play the major role in pathogenesis and clinical behaviour of the tumour. Aim To investigate the association of soluble interleukin 2α receptor (sIL-2Rα) and interleukin-2 (IL-2) serum concentrations with BCC. Material and methods The study involved 110 individuals with BCC and 60 healthy age- and sex-matched volunteers. Serum levels of sIL-2Rα and IL-2 were measured using ELISA test. Results We found significantly (p = 0.027) increased sIL-2Rα serum levels in BCC patients, in comparison to healthy controls. Statistically (p = 0.04) higher sIL-2Rα levels were observed in patients with more advanced tumours. Serum levels of sIL-2Rα showed a significant linear (r = 0.24, p = 0.018) correlation with tumour size. The average IL-2 serum levels in BCC patients were statistically (p = 0.039) decreased compared to controls. Significantly (p = 0.0454) lower median IL-2 levels were observed in patients with more advanced tumours. A negative correlation between sIL-2Rα and IL-2 serum concentrations was revealed (r = –0.22; p = 0.027). Conclusions Our results testify to the importance of the IL-2/sIL-2Rα signalling pathway in pathogenesis of BCC, suggesting that IL-2 and sIL-2Rα might be considered as potential markers of disease and targets for immunotherapy in BCC patients. PMID:27605896

  14. Modification of soluble immunological parameters during treatment with interleukin-2.

    PubMed

    Abbate, I; Correale, M; Musci, M D; Guida, M; Dragone, C D; De Santis, M; De Lena, M

    1993-01-01

    In the present study we tested numerous soluble immunological parameters (soluble Interleukin 2 receptor, Beta 2 microglobulin, Neopterin, soluble CD8 antigen, gamma Interferon and alpha Tumor Necrosis Factor) in sera obtained from 18 advanced cancer patients during subcutaneous treatment with recombinant Interleukin 2. The treatment cycles consisted of a dose of 9 x 10(6) IU/m2 twice daily for 2 days followed by 1.8 x 10(6) IU/m2 twice daily for 5 days/week during 6 weeks. Even before therapy, neoplastic patients had higher levels of soluble Interleukin 2 receptor than a group of healthy subjects. Moreover, basal soluble CD8 antigen levels showed significant differences (p < 0.01) in patients with different clinical responses (responders and non-responders). During treatment we observed a fast increase (in the first or second week) of all parameters considered; soluble Interleukin 2 receptor and soluble CD8 antigen were the markers that were best related to the course of immunotherapy. In conclusion, monitoring recombinant Interleukin 2 immunotherapy with immunological parameters in serum seems to be of interest. However, more data are necessary to confirm the real value of the single markers. PMID:8138662

  15. Regulation of the human interleukin-2/interleukin-2 receptor system: A role for immunosuppression

    SciTech Connect

    Kaempfer, R.

    1994-12-31

    The strength of the cellular immune response is regulated to a large extent by the amount of interleukin-2 (IL-2) produced in response to a stimulus. The ability of lymphocytes and other cells to respond to IL-2 depends upon the expression of cell surface IL-2 receptors. Formation of a high-affinity IL-2 receptor is regulated primarily through induction of its {alpha} subunit, IL-2R{alpha}. Once formed, the IL-2R{alpha} chain turns over rapidly, rendering expression of high-affinity IL-2 receptors during the immune response dependent upon continuous activity of the IL-2R{alpha} gene. The induced expression of both human IL-2 and IL-2R{alpha} chains is sensitive to cell-mediated suppression by CD8 cells; depletion of CD8 cells leads to extensive superinduction. This coupled suppression of IL-2 and IL-2R{alpha} genes greatly increases the extend of control, and strongly limits the strength, of the signal transduced by this ligand/receptor system during an immune response. 29 refs., 3 figs.

  16. Role of Human CD36 in Bacterial Recognition, Phagocytosis and Pathogen-Induced C-Jun N-Terminal Kinase (JNK) - Mediated Signaling 1

    PubMed Central

    Baranova, Irina N.; Kurlander, Roger; Bocharov, Alexander V.; Vishnyakova, Tatyana G.; Chen, Zhigang; Remaley, Alan T.; Csako, Gyorgy; Patterson, Amy P.; Eggerman, Thomas L.

    2013-01-01

    Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6-signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacteria, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both Gram-positive and Gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36-transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to Gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with Gram-positive bacteria and lipotechoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40%–50% in human fibroblasts induced by both Gram-negative and Gram-positive bacteria as well as LPS. Of all MAP kinase signaling cascade inhibitors tested, only the inhibitor of JNK, a stress activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-κB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an α-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by Gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner. PMID:18981136

  17. 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.

  18. Temporal sequence and cellular origin of interleukin-2 stimulated cytokine gene expression.

    PubMed Central

    Saraya, K. A.; Balkwill, F. R.

    1993-01-01

    A study of activation of the cytokine network by interleukin 2, IL-2, may provide a rationale for devising cytokine combination and cytokine antagonist treatments with increased anti-tumour efficacy and decreased toxicity. We have investigated the expression of mRNA for 13 cytokines and three transcription factors during in vitro culture of peripheral blood mononuclear cells, PBMC, with IL-2. A consistent pattern of induction was seen in nine individuals, with early (2-24 h) induction of IL-1 beta, IL-6, tumour necrosis factor, TNF, lymphotoxin, LT, and gro. TNF and LT mRNA was expressed continually throughout culture, but levels of mRNA for IL-1 beta, IL-6, and gro declined by 24-48 h. After 48 h, PBMC began to express mRNA for IFN-gamma, IL-5, GM-CSF, and M-CSF. At 15 min to 1 h post IL-2 mRNA for c-fos, c-jun, and c-myc, and TNF was induced in three individuals studied. IL-4, IFN-alpha, and IL-1 alpha mRNA was not detected. Only a minority of cells expressed mRNA for TNF, IL-1 beta, IL-6 and IFN-gamma, and monocytes were the main source. Levels of cytokine protein in culture supernatants mirrored the pattern of mRNA induction. This in vitro model shows clear parallels with the reported in vivo production of cytokines during IL-2 therapy, and may prove useful in designing new therapeutic strategies. Images Figure 1 Figure 2 Figure 3 PMID:8439502

  19. 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; Müller, 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

  20. Ginsenoside Rb3 protects cardiomyocytes against ischemia-reperfusion injury via the inhibition of JNK-mediated NF-κB pathway: a mouse cardiomyocyte model.

    PubMed

    Ma, Lijia; Liu, Huimin; 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-κB 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-κB signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-κB activity. Ginsenoside Rb3 inhibits the upregulation of phospho-IκB-α and nuclear translocation of NF-κB 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-α, 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-κB pathway. Finally, the upstream factors of NF-κB 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-κB 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-κB activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury. PMID

  1. Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-κB 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-κB 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-κB signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-κB activity. Ginsenoside Rb3 inhibits the upregulation of phospho-IκB-α and nuclear translocation of NF-κB 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-α, 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-κB pathway. Finally, the upstream factors of NF-κB 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-κB 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-κB activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury. PMID

  2. 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

  3. Interleukin-2 dependent cytotoxic T-cell clones

    SciTech Connect

    Palladino, M.

    1987-07-28

    A method is described of stimulating production of the lymphokines ..cap alpha..-interferon and ..beta..-interferon by interleukin-2 dependent cytotoxic cultured T-cell lines comprising administering to a T-cell line selected from the group consisting of T-cell lines CTLL-RP (CRL 8201), CTLL-R8 (CRL 8202), CTLL-R9 (CRL 8203), CTLL-R11 (CRL 8204), and CTLL-R12 (CRL 8205). An amount of an antigen selected from the group consists of Newcastle Disease Virus and Sendai Virus sufficient to cause stimulation of production of the lymphokines.

  4. 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.

  5. Continuous infusion interleukin-2 and antihistamines in metastatic kidney cancer.

    PubMed

    Walker, Paul R; Khuder, Sadik A; Quan, Walter D Y

    2005-10-01

    A prior randomized trial suggested a possible survival advantage favoring the combination of histamine and subcutaneous interleukin-2 (IL-2), compared to IL-2 alone in patients with metastatic melanoma. It has been postulated previously that antihistamines may, therefore, actually be antagonistic to IL-2 and thus interfere with its antitumor activity. We have previously shown no such antagonistic effect in patients with melanoma receiving IL-2 and antihistamines when reviewing the known literature. We sought to determine whether there was any negative effect of the combination in patients with metastatic kidney cancer. A PubMed literature search between 1985 and 2005 was done. High-dose continuous (or constant) infusion (CIV) interleukin-2 was used as the reference therapy because of the relatively constant IL-2 levels generated by this approach. Studies in which cimetidine, ranitidine, or famotidine were regularly scheduled and administered concurrently with IL-2 were included. Thirteen studies were identified. A total of 47 patients responded to therapy. Total response rate = 22%; 95%; Confidence Interval: 17%-28%. Eleven complete responses were noted. Complete response rate = 5%; 95% Confidence Interval: 3%-9%. These response rates are consistent with previously noted IL-2 response rates. In this study of CIV IL-2 and antihistamines, this combination appears to be active in metastatic kidney cancer. There appears to be no negative effect of antihistamine on the CIV IL-2 response rate in this disease. PMID:16248764

  6. 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

  7. Plasma soluble interleukin-2 receptor in patients with primary myelofibrosis.

    PubMed

    Wang, J C; Wang, A

    1994-02-01

    Using en enzyme-linked immunosorbent assay (ELISA) test, the level of soluble Tac peptide, one chain of the human interleukin-2 receptor, was measured in the plasma of 26 patients with primary myelofibrosis (MF), seven patients with polycythaemia vera and 11 normal controls. The plasma soluble interleukin-2 receptor (sIL-2R) was found to be significantly elevated in patients with primary MF compared to polycythaemia vera or controls (P < 0.001), while the plasma sIL-2R of patients with polycythaemia vera also was found to be significantly elevated compared to controls (P < 0.01). The significantly elevated value of sIL-2R seen in primary MF may be secondary to T cell activation resulting from autoimmune phenomena, and myeloblast activation with release of sIL-2R may also be a contributing factor. In primary MF, plasma sIL-2R levels were also found to be correlated to survival, circulating blast cell counts, and thrombocytopenia, but not to white blood cell counts, LDH levels, degree of marrow fibrosis, or degree of splenomegaly. Patients with primary MF with higher titre of plasma sIL-2R had a shorter survival. Further studies involving more patients and longer follow-up may substantiate that plasma sIL-2R is an important prognostic indicator in primary MF. PMID:8199029

  8. [Interleukin 2 revival: a revisited model and new therapeutic applications].

    PubMed

    Jacques, Yannick; Mortier, Erwan

    2016-01-01

    Interleukin-2, a cytokine identified as T-cell growth factor, has long been regarded as central to the development and effector activities of immune responses. Several gene knockout mouse studies and observations in humans, however, have undermined that vision, and the discovery of regulatory T cells showed that IL-2, in contrast to the accepted dogma, has the essential function of promoting (1) homeostasis and (2) the function of these T regulator cells the which, limit the action of the effector cells, in particular to prevent the autoimmune reaction drifts. This new paradigm has major implications on the use of IL-2 in therapy, and creates new strategies to manipulate the Teffectors/Tregulators balance. PMID:27406772

  9. Cloning, sequence, and expression of bovine interleukin 2.

    PubMed Central

    Cerretti, D P; McKereghan, K; Larsen, A; Cantrell, M A; Anderson, D; Gillis, S; Cosman, D; Baker, P E

    1986-01-01

    Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system. Images PMID:3517854

  10. Pure Red Cell Aplasia Following Interleukin-2 Therapy

    PubMed Central

    Dutcher, Janice P.; Fan, Wen; Wiernik, Peter H.

    2016-01-01

    A 61-year-old woman with metastatic renal cell carcinoma underwent systemic treatment with high-dose interleukin-2 (IL-2). Anemia requiring transfusion of 1 unit of packed red blood cells (PRBCs) was required during the second week of IL-2 therapy. One month following completion of high-dose IL-2 treatment, she was hospitalized for severe, symptomatic anemia and received 5 units of PRBCs. She was referred back for evaluation. A complete hematologic evaluation was performed including antiviral serology, evaluation for hemolysis, complete iron studies, and finally bone marrow aspiration and biopsy. The diagnosis was pure red cell aplasia, and no inciting viral cause could be ascertained. She required PRBCs for 5 months following IL-2 therapy. It was concluded that IL-2 was the cause of her red cell aplasia. This subsequently resolved spontaneously, and she had normal hemoglobin and hematocrit, respectively, 1 and 2 years after treatment. PMID:27144182

  11. 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.

  12. Type I Diabetes-Associated Tolerogenic Properties of Interleukin-2

    PubMed Central

    Chentoufi, Aziz Alami; Gaudreau, Simon; Nguyen, Alex; Sabha, Mahmoud; Amrani, Abdelaziz; ElGhazali, Geyhad

    2011-01-01

    Type 1 Diabetes (T1D) results from insulin-producing beta cells destruction by diabetogenic T lymphocytes in humans and nonobese diabetic (NOD) mice. The breakdown of tolerance has been associated with a defect in the number and the function of naturally occurring regulatory T cells (nTreg) that are the master player in peripheral tolerance. Gene knockout experiments in mouse models have shown a nonredundant activity of IL-2 related to its critical role in inducing nTreg and controlling peripheral T cell tolerance. Whereas strong evidence has suggested that IL-2 is critically required for nTreg-mediated T1D control, several fundamental questions remain to be addressed. In this paper, we highlight the recent findings and controversies regarding the tolerogenic properties of IL-2 mediated through nTreg. We further discuss a potential link between the immunomodulatory role of interleukin-2 and the pathogenesis of type 1 diabetes. PMID:21647403

  13. Enhancement of interleukin-2 immunotherapy with L-arginine.

    PubMed Central

    Lieberman, M D; Nishioka, K; Redmond, H P; Daly, J M

    1992-01-01

    Nutrient substrates have been shown to enhance cell-mediated immunity, but their role as adjuvants to immunotherapy has not been previously determined. This study evaluated L-arginine as an essential substrate for optimal generation of lymphokine-activated killer (LAK) cells. This experiment also assessed supplemental dietary L-arginine as a means to potentiate the host antitumor response to interleukin-2 (IL-2) in a murine neuroblastoma (NRB) model. A/J mice received 1% arginine or isonitrogenous 1.7% glycine in addition to a regular diet 14 days before subcutaneous inoculation with C1300 NRB cells. Twenty-four hours later, animals received low (1 x 10(6) U/kg three times a day) or high (3 x 10(6) U/kg three times a day) doses of IL-2 or saline intraperitoneally for 4 days. On days 4 and 10 post-C1300 NRB inoculation, mice were killed for assessment of natural killer cell and tumor specific cytotoxicity. Remaining animals were followed for tumor incidence, tumor growth, and duration of host survival. Interleukin-2 therapy in mice receiving dietary arginine compared with those receiving glycine resulted in significantly augmented natural killer cell cytotoxicity (day 4) and generation of specific tumoricidal mechanisms (day 10). The addition of dietary arginine to low-dose IL-2 therapy significantly diminished C1300 NRB engraftment (p less than 0.05) and growth (p less than 0.001) and prolonged the duration of host survival (p less than 0.05) compared with the glycine treatment group. In vitro studies demonstrated that L-arginine is an essential substrate for optimal generation of LAK cells. Thus, supplemental dietary L-arginine enhances lymphocyte cytotoxic mechanisms and potentiates IL-2 immunotherapy. PMID:1546902

  14. Human Interleukin-2 and Hen Egg White Lysozyme: Screening for Bacteriolytic Activity against Various Bacterial Cells

    PubMed Central

    Levashov, P. A.; Ovchinnikova, E. D.; Morozova, O. A.; Matolygina, D. A.; Osipova, H. E.; Cherdyntseva, T. A.; Savin, S. S.; Zakharova, G. S.; Alekseeva, A. A.; Belogurova, N. G.; Smirnov, S. A.; Tishkov, V. I.; Levashov, A. V.

    2016-01-01

    The bacteriolytic activity of interleukin-2 and hen egg white lysozyme against 34 different species of microorganisms has been studied. It was found that 6 species of microorganisms are lysed in the presence of interleukin-2. All interleukin-2-sensitive microorganisms belong either to the Enterobacteriaceae, Bacillaceae, or the Lactobacillaceae family. It was also found that 12 species of microorganisms are lysed in the presence of lysozyme, and 16 species of microorganisms are lysed in the presence of sodium dodecyl sulfate (SDS). The bacteriolytic activity of interleukin-2 and lysozyme was studied at various pH values. PMID:27099789

  15. Continuous infusion interleukin-2 and intravenous famotidine in metastatic melanoma.

    PubMed

    Quan, Walter D Y; Milligan, Karen S; Quan, Francine M; Cuenca, Rosa E; Khan, Nawazish; Liles, Darla K; Walker, Paul R

    2006-12-01

    Lymphokine-activated killer cell (LAK) cytotoxicity against tumor cells is induced by the use of high-dose infusional interleukin-2 (IL-2). LAK cytotoxicity against neoplastic cells may be augmented by famotidine. Twelve (12) patients have been treated with continuous infusion IL-2 (18 MIU/m2/24 hours) for 72 hours and famotidine 20 mg IVPB twice per day. Cycles were repeated every 3 weeks. These patients were of median age--67 years (range, 25-79), had a median performance status of 1 (range, 0-1), and had metastatic sites, including lung, lymph node, subcutaneous/soft tissue, and liver. The most common toxicities of this regimen were fever, rigors, nausea/emesis, hypophosphatemia, and hypomagnesemia. Three (3) partial responses have been seen (25% response rate). One (1) of these responders has undergone complete surgical resection and is disease-free at 15+ months. Four (4) patients are alive at a median of > 25 months. The median survival for all patients is 13 months. This combination of infusional IL-2 with famotidine is active in metastatic melanoma. PMID:17257076

  16. Continuous infusion interleukin-2 and famotidine in metastatic kidney cancer.

    PubMed

    Quan, Walter D Y; Vinogradov, Mikhail; Quan, Francine M; Khan, Nawazish; Liles, Darla K; Walker, Paul R

    2006-10-01

    Infusional interleukin-2 (IL-2) is able to elicit lymphokine-activated killer cell (LAK) cytotoxicity against kidney cancer in vitro and in vivo. Famotidine may be able to augment LAK cytotoxicity against neoplastic cells. Fifteen (15) patients were treated with continuous-infusion IL-2 (9-18 MIU/m2/24 hours) for 72 hours and famotidine 20 mg intravenously twice per day. Cycles were repeated every 3 weeks. These patients had a median age of 60 years (range, 29-72), had a median performance status of 1 (range, 0-1), and had metastatic sites, including lung, bone, lymph node, and liver. The most common toxicities of this regimen were hypophosphatemia, fever, nausea/emesis, rigors, elevated creatinine, and hypomagnesemia. One (1) complete and 6 partial responses have been seen (47% response rate). The median duration of response is 9 months. The median survival for all patients is 20 months. Five (5) patients are alive at a median of 36+ months. This combination of infusional IL-2 with famotidine is active in metastatic kidney cancer. PMID:17105423

  17. Soluble serum interleukin 2 receptor levels in leprosy patients

    PubMed Central

    Tung, K. S. K.; Umland, Edith; Matzner, P.; Nelson, K.; Schauf, Victoria; Rubin, L.; Wagner, D.; Scollard, D.; Vithayasai, Prakong; Vithayasai, Vicharn; Worobec, Sophie; Smith, T.; Suriyanond, Vinai

    1987-01-01

    Soluble interleukin 2 receptors (IL-2R) in sera of leprosy patients from Chiang Mai, Thailand, were quantified with a solid phase enzyme immunoassay using two monoclonal antibodies to the IL-2R. The IL-2R levels of untreated lepromatous, borderline lepromatous or midborderline patients and treated lepromatous and borderline lepromatous or treated borderline tuberculoid and tuberculoid patients were comparable to those of the Thai household or nonhousehold contacts; and they were significantly higher than the levels of USA control subjects. In contrast, IL-2R of untreated tuberculoid or borderline tuberculoid patients were significantly reduced. Patients with ongoing reversal reaction had very high circulating IL-2R, the levels of which correlated with fever and extent of skin lesions. Although erythrema nodosum leprosum patients also had elevated IL-2R levels, they were significantly below those of patients with reversal reaction. When treated with corticosteroid, precipitous reduction of IL-2R was noted in all patients with reversal reaction but not in patients with erythema nodosum leprosum. PMID:3115652

  18. Modeling interleukin-2-based immunotherapy in AIDS pathogenesis.

    PubMed

    Joly, Marcel; Odloak, Darci

    2013-10-21

    In this paper, we sought to identify the CD4(+) T-cell dynamics in the course of HIV infection in response to continuous and intermittent intravenous courses of interleukin-2 (IL-2), the principal cytokine responsible for progression of CD4(+) T-lymphocytes from the G1 to the S phase of the cell cycle. Based on multivariate regression models, previous literature has concluded that the increase in survival of CD4(+) T-cell appears to be the critical mechanism leading to sustained CD4(+) T-cell levels in HIV-infected patients receiving intermittent IL-2 therapy. Underscored by comprehensive mathematical modeling, a major finding of the present work is related to the fact that, rather than due to any increase in survival of CD4(+) T-cells, the expressive, selective and sustained CD4(+) T-cell expansions following IL-2 administration may be related to the role of IL-2 in modulating the dynamics of Fas-dependent apoptotic pathways, such as activation-induced cell death (AICD) or HIV-specific apoptotic routes triggered by viral proteins. PMID:23806696

  19. Interleukin 2 maintains biologic stability and sterility over prolonged time.

    PubMed

    Safar, M; Junghans, R P

    2000-09-01

    The FDA approved interleukin 2 (IL2) for clinical use in 1992 in a high-dose bolus intravenous infusion schedule. IL2 administered by continuous low- and intermediate-dose infusion can result in a variety of immunologic effects including the expansion of the Natural Killer (NK) cell pool and immune reconstitution in immune-deficient hosts. These immune modifications are essential for augmentation of both currently available and evolving immunotherapies. The manufacturer's data indicate stability of the IL2 for a period of 6 days. This time frame is not practical for prolonged infusional schemes necessitating frequent changes of drug depots. We tested the biologic stability and sterility of the commercially available recombinant IL2 preparation (aldesleukin; Proleukin, Chiron) under clinical conditions for up to 30 days. Our results confirm that IL2 retains its biologic activity and sterility under these conditions for prolonged periods. This information will simplify IL2 outpatient regimens, allowing for convenient intervals for drug depot renewal, leading to improved patient compliance and conserved health care expenditures. PMID:10996039

  20. 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

  1. 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.

  2. [Biological properties and therapeutic use of interleukin 2 (IL-2)].

    PubMed

    Robak, T

    1995-01-01

    A cytokine produced by the subpopulation of activated helper lymphocytes T has been called interleukin-2 (IL-2). The obtaining of recombinant cytokine has facilitated the study of its biological properties and its application in the treatment of certain neoplastic and infectious diseases. IL-2 affects the target cells by means of a receptor of great affinity consisting of three independent chains: alpha, beta, gamma. The cytokine is the most important growth factor of lymphocytes T, conditioning their clonal expansion. Antigen stimulation is the condition for the expression of IL-2 does not, however, affect resting lymphocytes T. The expression of the receptor for this cytokine on NK cells is, however, continuous in character but only a very small percentage of these cells has receptors of great affinity. IL-2 plays a great role in adoptive immunotherapy consisting in intravenous administration of cells with cytotoxic properties. Cells obtained from peripheral blood and grown in vitro are called LAK cells (lymphocyte activated killer cells), while cells obtained from neoplasms and grown in similar conditions are named TIL cells (tumor infiltrated lymphocytes). LAK and TIL cells reveal a similar antineoplastic activity in vivo. At present, however, recombinant IL-2 alone is used more often, either intravenously or subcutaneously. The cytokine is effective in the treatment of patients with disseminate cancer of the kidney and melanoma, and in adjuvant therapy of acute myeloid leukemia. Attempts have been made to apply it in the treatment of AIDS and leprosy. The toxic effect of IL-2 depends on the dose and the mode of administration. In the majority of patients parainfluenza symptoms appear. Most undesirable effects are connected with multisystemic syndrome of capillary vessels hyperpermeability leading to the increased fluid retention into extravascular spaces, oedema, hypotonia and oliguria. PMID:8657637

  3. Increasing Regulatory T Cells With Interleukin-2 and Interleukin-2 Antibody Complexes Attenuates Lung Inflammation and Heart Failure Progression.

    PubMed

    Wang, Huan; Hou, Lei; Kwak, Dongmin; Fassett, John; Xu, Xin; Chen, Angela; Chen, Wei; Blazar, Bruce R; Xu, Yawei; Hall, Jennifer L; Ge, Jun-Bo; Bache, Robert J; Chen, Yingjie

    2016-07-01

    Congestive heart failure (CHF) is associated with an increase of leukocyte infiltration, proinflammatory cytokines, and fibrosis in the heart and lung. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) suppress inflammatory responses in various clinical conditions. We postulated that expansion of Tregs attenuates CHF progression by reducing cardiac and lung inflammation. We investigated the effects of interleukin-2 (IL-2) plus IL-2 monoclonal antibody clone JES6-1 complexes (IL2/JES6-1) on induction of Tregs, transverse aortic constriction-induced cardiac and lung inflammation, and CHF progression in mice. We demonstrated that end-stage CHF caused a massive increase of lung macrophages and T cells, as well as relatively mild left ventricular (LV) leukocyte infiltration. Administration of IL2/JES6-1 caused an ≈6-fold increase of Tregs within CD4(+) T cells in the spleen, lung, and heart of mice. IL2/JES6-1 treatment of mice with existing transverse aortic constriction-induced LV failure markedly reduced lung and right ventricular weight and improved LV ejection fraction and LV end-diastolic pressure. Mechanistically, IL2/JES6-1 treatment significantly increased Tregs; suppressed CD4(+) T-cell accumulation; dramatically attenuated leukocyte infiltration, including decreasing CD45(+) cells, macrophages, CD8(+) T cells, and effector memory CD8(+); and reduced proinflammatory cytokine expressions and fibrosis in the lung of mice. Furthermore, IL2/JES6-1 administered before transverse aortic constriction attenuated the development of LV hypertrophy and dysfunction in mice. Our data indicate that increasing Tregs through administration of IL2/JES6-1 effectively attenuates pulmonary inflammation, right ventricular hypertrophy, and further LV dysfunction in mice with existing LV failure, suggesting that strategies to properly expand Tregs may be useful in reducing CHF progression. PMID:27160197

  4. Interleukin-2/Anti-Interleukin-2 Immune Complex Expands Regulatory T Cells and Reduces Angiotensin II-Induced Aortic Stiffening.

    PubMed

    Majeed, Beenish; Tawinwung, Supannikar; Eberson, Lance S; Secomb, Timothy W; Larmonier, Nicolas; Larson, Douglas F

    2014-01-01

    Adaptive immune function is implicated in the pathogenesis of vascular disease. Inhibition of T-lymphocyte function has been shown to reduce hypertension, target-organ damage, and vascular stiffness. To study the role of immune inhibitory cells, CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), on vascular stiffness, we stimulated the proliferation of Treg lymphocytes in vivo using a novel cytokine immune complex of Interleukin-2 (IL-2) and anti-IL-2 monoclonal antibody clone JES6-1 (mAbCD25). Three-month-old male C57BL/6J mice were treated with IL-2/mAbCD25 concomitantly with continuous infusion of angiotensin type 1 receptor agonist, [Val(5)]angiotensin II. Our results indicate that the IL-2/mAbCD25 complex effectively induced Treg phenotype expansion by 5-fold in the spleens with minimal effects on total CD4(+) and CD8(+) T-lymphocyte numbers. The IL-2/mAbCD25 complex inhibited angiotensin II-mediated aortic collagen remodeling and the resulting stiffening, analyzed with in vivo pulse wave velocity and effective Young's modulus. Furthermore, the IL-2/mAbCD25 complex suppressed angiotensin II-mediated Th17 responses in the lymphoid organs and reduced gene expression of IL-17 as well as T cell and macrophage infiltrates in the aortic tissue. This study provides data that support the protective roles of Tregs in vascular stiffening and highlights the use of the IL-2/mAbCD25 complex as a new potential therapy in angiotensin II-related vascular diseases. PMID:25258681

  5. ent-kaurane diterpenoids from Croton tonkinensis induce apoptosis in colorectal cancer cells through the phosphorylation of JNK mediated by reactive oxygen species and dual-specificity JNK kinase MKK4.

    PubMed

    Thuong, Phuong Thien; Khoi, Nguyen Minh; Ohta, Saho; Shiota, Shinichiro; Kanta, Hironori; Takeuchi, Kenji; Ito, Fumiaki

    2014-01-01

    To search for new chemotherapeutic agents to treat colorectal cancer, we isolated a number of natural ent-kaurane diterpenoids from the plant Croton tonkinensis. Among them, only CeKDs with the 15-oxo-16-ene moiety induced the apoptosis of colorectal cancer cell lines Caco-2 and LS180. The active CeKD induced the activation of ERK and JNK, but the inactive ones induced that of ERK, but not that of JNK. It thus appears that JNK seemed to play an important role in the apoptotic activity of the active compounds. The dualspecificity JNK kinase MKK4 was activated in both colorectal cancer cells treated with the active CeKD, but MKK7 was not activated. Further, the active CeKD, but not the inactive one, enhanced the generation of intracellular reactive oxygen species (ROS) in both cells. CeKD-induced cell apoptosis and ROS generation, as well as JNK activation, were inhibited by the antioxidant N-acetyl-L-cysteine. These findings suggest that ROS stimulated the phosphorylation of JNK mediated by MKK4 and played a critical role in CeKD-induced apoptosis in colorectal cancer cells. PMID:24476312

  6. 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.

  7. 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

  8. A randomized phase II trial of interleukin 2 and interleukin 2-interferon alpha in advanced renal cancer.

    PubMed Central

    Jayson, G. C.; Middleton, M.; Lee, S. M.; Ashcroft, L.; Thatcher, N.

    1998-01-01

    A randomized phase II trial was performed to compare the efficacy and toxicity of interleukin 2 (IL-2) with an IL-2 and interferon alpha (IFN-alpha) regimen for the treatment of metastatic renal carcinoma. Sixty patients with recurrent renal cell carcinoma (RCC) who had previously undergone a nephrectomy were randomized to receive three cycles of IL-2 or IL-2 with IFN-alpha2b. Eighteen MU of IL-2 were administered subcutaneously on Mondays-Fridays for 3 weeks out of 4. Those patients randomized to receive the combination received the same regimen of IL-2 with 9 MU of IFN-alpha2b subcutaneously on Mondays, Wednesdays and Fridays for 3 weeks out of 4. Thirty patients were randomized to receive each arm. Twenty-nine were evaluable in each arm. Twenty-two patients received three cycles of IL-2 but only 14 patients received three cycles of IL-2/IFN-alpha because of the greater toxicity of the combination. The principal toxicities included nausea, fatigue and fever. There were no complete responses in either arm and only two patients who were treated with IL-2 attained a partial response. Twelve patients in each arm had stable disease and 15 patients in the IL-2 arm and 16 patients in the IL-2/IFN-alpha arm progressed through treatment. There were no significant differences in survival. Ten patients who received IL-2 are alive with a median follow-up of 266 days, whereas six patients who received IL-2/IFN-alpha are alive after a median of 278 days. The median survival from the time of identification of metastatic disease is 444 days in the IL-2 arm and 381 days in the IL-2/IFN-alpha arm. The IL-2/IFN-alpha combination is more toxic than IL-2 alone and this resulted in a reduced number of cycles of treatment. However, the median survival of the two groups was the same, suggesting that further evaluation of the IL-2/IFN-alpha combination should be confined to large prospective randomized clinical trials. PMID:9703284

  9. Chemokine gene expression in the murine renal cell carcinoma, RENCA, following treatment in vivo with interferon-alpha and interleukin-2.

    PubMed Central

    Sonouchi, K.; Hamilton, T. A.; Tannenbaum, C. S.; Tubbs, R. R.; Bukowski, R.; Finke, J. H.

    1994-01-01

    The expression of three chemoattractant cytokine (chemokine) messenger (m)RNAs in the murine renal cell carcinoma (RENCA) from mice treated with a combination of interferon-alpha (IFN-alpha) and interleukin-2 was examined and related to tumor infiltration by inflammatory leukocytes. Using a semi-quantitative reverse transcriptase polymerase chain reaction assay, mRNAs encoding the KC, JE, and IP-10 genes were all elevated in tumor tissue from mice treated systemically with IFN-alpha/interleukin-2 for 4 days. Similarly, the mRNA for tumor necrosis factor-alpha (TNF-alpha) was also increased in tumors from treated as compared to control animals. The same tumors showed a significant increase in Mac-1+ leukocytes, which correlated well with the increase in chemokine and TNF-alpha gene expression. The renal cell carcinoma tumor itself may be responsible for the expression of chemokine genes in the tumor bed following cytokine therapy. Cultures of freshly explanted RENCA cells expressed significant levels of chemokine mRNAs when stimulated in vitro with IFN alpha, IFN gamma, and/or interleukin-2, demonstrating that this tumor cell has potential for expression of these genes in vivo. In contrast, TNF-alpha expression was not detected in cultured tumor cells. Thus TNF-alpha may be expressed by infiltrating monocytes following exposure to recombinant cytokine therapy. Images Figure 1 Figure 2 Figure 4 PMID:8160774

  10. 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.

  11. Molecular mechanism of interleukin-2-induced mucosal homeostasis.

    PubMed

    Mishra, Jayshree; Waters, Christopher M; Kumar, Narendra

    2012-03-01

    Sustained damage to the mucosal lining in patients with inflammatory bowel disease (IBD) facilitates translocation of intestinal microbes to submucosal immune cells leading to chronic inflammation. Previously, we demonstrated the role of Jak3 in IL-2-induced intestinal epithelial cell (IEC) migration, one of the early events during intestinal wound repair. In this study, we demonstrate that IL-2 also plays a role in IEC homeostasis through concentration-dependent regulation of IEC proliferation and cell death. At lower concentrations (≤50 U/ml), IL-2 promoted proliferation, while at higher concentrations (100 U/ml), it promoted apoptosis. Activation by IL-2 led to tyrosine phosphorylation-dependent interactions between Jak3 and p52ShcA only at lower concentrations. Phosphatase SHP1 dephosphorylated IL-2-induced phosphorylated p52ShcA. Higher concentrations of IL-2 decreased the phosphorylation of Jak3 and p52ShcA, disrupted their interactions, redistributed Jak3 to the nucleus, and induced apoptosis in IEC. IL-2 also induced dose-dependent upregulation of p52shcA and downregulation of jak3-mRNA. Constitutive overexpression and mir-shRNA-mediated knockdown studies showed that expression of both Jak3 and p52ShcA were necessary for IL-2-induced proliferation of IEC. Doxycycline-regulated sh-RNA expression demonstrated that IL-2-induced downregulation of jak3-mRNA was responsible for higher IL-2-induced apoptosis in IEC. Collectively, these data demonstrate a novel mechanism of IL-2-induced mucosal homeostasis through posttranslational and transcriptional regulation of Jak3 and p52ShcA. PMID:22116305

  12. 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.

  13. Differential gene expression in response to adjunctive recombinant human interleukin-2 immunotherapy in multidrug-resistant tuberculosis patients.

    PubMed

    Johnson, B J; Estrada, I; Shen, Z; Ress, S; Willcox, P; Colston, M J; Kaplan, G

    1998-06-01

    Administration of low-dose recombinant human interleukin 2 (rhuIL-2) in combination with multidrug chemotherapy to patients with multidrug-resistant tuberculosis (MDR TB) induces measurable changes in in vitro immune response parameters which are associated with changes in the clinical and bacteriologic status of the patients. To determine the molecular basis of these changes, we have used semiquantitative reverse transcriptase-initiated PCR (RT-PCR) and differential display technology. During rhuIL-2 treatment of MDR TB patients, decreased levels of gamma interferon (IFN-gamma) mRNA in peripheral blood mononuclear cells (PBMC) relative to baseline levels were observed. However, at the site of a delayed-type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD), the expression of cellular IFN-gamma and IL-2 mRNAs was increased during rhuIL-2 therapy. Levels of other cytokine mRNAs were not significantly affected by rhuIL-2 administration. Using differential-display RT-PCR, we identified several genes expressed at the DTH skin test site which were up- or down-regulated during rhuIL-2 treatment. Cytochrome oxidase type I mRNA was increased in response to rhuIL-2 therapy relative to baseline levels, as was heterogeneous nuclear ribonuclear protein G mRNA. CD63, clathrin heavy chain, and beta-adaptin mRNAs, all of which encode proteins associated with the endocytic vacuolar pathway of cells, were also differentially regulated by rhuIL-2 administration. The differential effects of IL-2 were confirmed in vitro by using PBMC obtained from PPD-positive individuals stimulated with Mycobacterium tuberculosis and IL-2. The differential expression of genes may provide a surrogate marker for leukocyte activation at a mycobacterial antigen-specific response site and for the development of an enhanced antimicrobial response which may result in improved outcomes in MDR TB patients. PMID:9596698

  14. Interleukin-2 treatment of tumor patients can expand regulatory T cells

    PubMed Central

    Beyer, Marc

    2012-01-01

    Augmented numbers of regulatory T cells contribute to the overall immunosuppression in tumor patients. Interleukin-2 has been widely used in the clinics in anticancer therapy, yet evidence has accumulated that the major drawback, limiting clinical efficacy, is the expansion of regulatory T cells, which aggravates immunosuppression. PMID:23170272

  15. Administration of high-dose interleukin-2 in a 2-year-old with metastatic melanoma.

    PubMed

    Bernhardt, M Brooke; Hicks, M John; Pappo, Alberto S

    2009-12-15

    Malignant melanoma is rare in pediatrics, and therapies for patients with disseminated disease have not been well studied. This report describes our experience with the use of high-dose interleukin 2 (aldesleukin, IL-2) in a 2-year-old child with metastatic melanoma and describes our approach for the administration of this agent to young patients. PMID:19731326

  16. 77 FR 22283 - Availability of an Environmental Assessment for Field Testing Feline Interleukin-2...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-13

    ...We are advising the public that the Animal and Plant Health Inspection Service has prepared an environmental assessment concerning authorization to ship for the purpose of field testing, and then to field test, an unlicensed Feline Interleukin-2 Immunomodulator, Live Canarypox Vector. The environmental assessment, which is based on a risk analysis prepared to assess the risks associated with......

  17. Repeated cycles with 72-hour continuous infusion interleukin-2 in kidney cancer and melanoma.

    PubMed

    Quan, Walter; Brick, Wendy; Vinogradov, Mikhail; Taylor, W Chris; Khan, Nawazish; Burgess, Russell

    2004-06-01

    While high-dose bolus inpatient interleukin-2 is generally given on 8-week cycles, continuous infusion interleukin-2 could potentially allow for more rapidly repeated cycles. Fourteen (14) patients with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, having either kidney cancer (6) or melanoma (8), have been treated with continuous infusion (CIV) interleukin-2 (IL-2) 18 MIU/m(2)/24 hours for 72 hours. Cycles were repeated every 3 weeks up to 4 cycles, then every 3-4 weeks for 2 cycles, then every 6-8 weeks, until progression or intolerable toxicity. All patients received famotidine 20 mg intravenously (i.v.) twice per day during the 72-hour infusions. Patient characteristics included a median ECOG performance status of 1; median age = 63 (range: 25-79); most common metastatic sites: lung (9), bone (5), lymph nodes (5), and the liver (3). No patients with metastatic kidney cancer underwent a nephrectomy prior to interleukin-2. Median number of cycles received = 5 (1-9). No patients required Intensive Care Unit (ICU) admission. There have been no treatment-related deaths. Most common toxicities have been rigors, fever, nausea/emesis, and the reversible elevation of creatinine. One complete response and three partial responses (67% response rate; 95% confidence interval: 30%-90%) have been seen in kidney cancer, and two partial responses (25% response rate; 95% confidence interval: 7%-60%) have occurred in melanoma. Median survival has not been reached at >9+ months. Responding sites include the liver, bone, lung, lymph node and subcutaneous sites. Inpatient 72-hour continuous infusion interleukin-2 at this dose and schedule is well tolerated by patients with an ECOG performance status of 0 or 1 and has activity in kidney cancer and melanoma. PMID:15285881

  18. 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

  19. Influence of human T-cell leukemia virus type I tax and rex on interleukin-2 gene expression.

    PubMed Central

    McGuire, K L; Curtiss, V E; Larson, E L; Haseltine, W A

    1993-01-01

    The X region of human T-cell leukemia virus type I (HTLV-I) encodes two proteins that regulate viral gene expression. The tax protein is the product of the transactivator gene and has been shown to up-regulate the expression of some cellular genes controlling T-cell replication, including that of the interleukin-2 (IL-2) T-cell growth hormone and the alpha chain of its receptor (IL-2R). Several studies have shown that tax transactivation of the IL-2R alpha-chain promoter is mediated by binding sites for the transcriptional activator NF-kappa B, and this mechanism has also been implicated in the tax activation of IL-2 promoter activity. The rex gene product of HTLV-I regulates viral protein production by influencing mRNA expression and has been implicated in the stabilization of IL-2R alpha-chain mRNA. In the present studies, the ability of the tax and rex proteins to transactivate IL-2 gene expression has been reinvestigated. The ability of the tax protein to transactivate IL-2 promoter activity appears, at least in part, to be mediated by the recognition sequence for a DNA-binding complex known as CD28RC. Consistent with this hypothesis is the observation that tax-mediated activation of IL-2 gene expression is resistant to the immunosuppressive affects of cyclosporin A, a property postulated for the CD28RC binding complex. Unexpectedly, this tax-mediated up-regulation of IL-2 expression is synergized by the presence of the rex protein. These findings demonstrate that transactivation of IL-2 gene expression by tax is augmented by mechanisms distinct from NF-kappa B and raise the possibility that rex, as well as tax, contributes to the oncogenic capability of HTLV-I by altering the expression of the IL-2 gene in T cells infected with this retrovirus. Images PMID:8382312

  20. Human Immune Disorder Arising from Mutation of the α 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 α 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.

  1. Focal takotsubo cardiomyopathy with high-dose interleukin-2 therapy for malignant melanoma.

    PubMed

    Damodaran, Senthil; Mrozek, Ewa; Liebner, David; Kendra, Kari

    2014-12-01

    High-dose interleukin-2 (IL-2) is an available treatment option for patients with metastatic melanoma or renal cell carcinoma, and is associated with sustained complete and partial responses in a subset of patients. IL-2, however, is not devoid of toxicities, most of which involve the cardiovascular system and manifest as hypotension, arrhythmias, and cardiomyopathy. This report describes an unusual presentation of takotsubo cardiomyopathy in a postmenopausal woman receiving high-dose IL-2 for metastatic melanoma. PMID:25505207

  2. Interleukin 2 production in a family with systemic lupus erythematosus and a C4Q0 heterozygous inheritance.

    PubMed Central

    Gutierrez, C; Cabrero, E; Vicario, J L; Martín Villa, M; Rengel, M A; Gomez Campdera, F J; Yebra, M; Fernández-Cruz, E; Arnaiz Villena, A

    1991-01-01

    Interleukin 2 production was studied in a family with systemic lupus erythematosus (SLE) and a C4Q0 heterozygous inheritance. Autoimmune manifestations seemed to be associated with the HLA haplotype containing the C4Q0 allele, which was shared by all four ill family members. Concentrations of interleukin 2, however, did not associate either with the haplotype or with the clinical or serological manifestations, as diminished concentrations of interleukin 2 were found in only two subjects with SLE. Thus the defect in this family seemed to be acquired rather than genetically conditioned. PMID:1888202

  3. Inhibition of G-Protein βγ Signaling Enhances T Cell Receptor-Stimulated Interleukin 2 Transcription in CD4+ T Helper Cells

    PubMed Central

    Yost, Evan A.; Hynes, Thomas R.; Hartle, Cassandra M.; Ott, Braden J.; Berlot, Catherine H.

    2015-01-01

    G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases. PMID:25629163

  4. 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

  5. Immunohistochemical correlates of response to recombinant interleukin-2-based immunotherapy in humans.

    PubMed

    Rubin, J T; Elwood, L J; Rosenberg, S A; Lotze, M T

    1989-12-15

    We have evaluated immunohistochemical characteristics of tumors and the infiltrating cells in patients treated with various immunotherapy regimens. Forty-eight patients with advanced malignancies were treated with high dose i.v. recombinant interleukin-2 alone or in combination with cyclophosphamide, recombinant tumor necrosis factor, recombinant interferon-alpha, antimelanoma antibody 9.2.27, adoptively transferred tumor infiltrating lymphocytes, or lymphokine-activated killer cells. Thirty-four patients with metastatic melanoma and two patients with breast carcinoma underwent excision of one or more s.c. metastases either before, during, or after treatment. Twelve patients with metastatic renal cell carcinoma underwent pretreatment nephrectomy and these tumors were also studied. Tumor cells were evaluated for class I (HLA-A,B,C) and II (HLA-DR) antigen expression and the mononuclear infiltrate was characterized using an avidin-biotin immunoperoxidase technique. All melanomas were class I antigen positive. Fifty-three % of biopsied metastatic melanoma lesions, 58% of primary renal cell carcinomas, and neither of the two breast carcinomas expressed class II antigen prior to therapy. The pretreatment expression of class II antigens by a tumor was not predictive of a clinical response to recombinant interleukin 2-based therapy. After treatment, however, seven of seven biopsied regressing individual metastases intensely expressed DR antigen on over fifty percent of the cells while only three of ten nonresponding lesions did so. Regressing lesions were permeated with macrophages and both CD4 and CD8 T-cell subsets. There were no CD1 or NKH-1 positive infiltrating cells detected in any lesion. The response to recombinant interleukin 2-based immunotherapy is associated with T-cell as well as macrophage infiltration. DR antigen expression by tumor cells and T-cell infiltrate appear in individual lesions to be associated with this response. PMID:2582450

  6. Interleukin-2 and syngeneic bone marrow transplantation in a murine fibrosarcoma model.

    PubMed

    Ho, S P; Stebler, B; Ershler, W B

    1991-04-01

    Mice received interleukin-2 (IL-2) either before and after, or just after intravenous inoculation of syngeneic fibrosarcoma cells. Fewer pulmonary tumor colonies were observed in those animals treated with IL-2, and the best results were observed when IL-2 was administered prior to tumor inoculation. When mice were lethally irradiated and reconstituted with tumor-contaminated bone marrow, IL-2 treatment was also associated with fewer tumor lung colonies. IL-2 may prove to be a useful adjuvant therapy, particularly in the setting of autologous bone marrow transplantation when the infused marrow is contaminated with tumor cells. PMID:1873353

  7. Hypersensitivity to aldesleukin (interleukin-2 and proleukin) presenting as facial angioedema and erythema.

    PubMed

    Abraham, Daryn; McGrath, Kris G

    2003-01-01

    Aldesleukin is a human recombinant interleukin-2 product. It also is known as interlukin-2 and Proleukin in the United States. It is indicated for the treatment of adults with metastatic renal cell carcinoma as well as for adults with metastatic melanoma. However, its use has been limited because of severe systemic toxicity. There have been no reports of aldesleukin producing a hypersensitivity reaction. This is the first reported case of an immediate systemic hypersensitivity reaction occurring after aldesleukin administration confirmed by enzyme-linked immunosorbent assay for specific immunoglobulin E against aldesleukin. PMID:12974198

  8. In vivo administration of interleukin-2 protects susceptible mice from Theiler's virus persistence.

    PubMed Central

    Larsson-Sciard, E L; Dethlefs, S; Brahic, M

    1997-01-01

    In vivo administration of interleukin-2 (IL-2)-secreting tumor cells results in complete protection against persistent infection by Theiler's murine encephalomyelitis virus (TMEV) in susceptible DBA/2 mice. The IL-2-mediated protection was found to depend on the inoculum size as well as the timing of IL-2 administration. IL-2-treated and TMEV-infected mice displayed a three- to fourfold relative increase in virus-specific cytotoxic T-lymphocyte (CTL) precursors. Thus, we postulate that the persistence of TMEV infection in susceptible mice reflects limited numbers of relevant CTL precursors and their time course of induction and activation. PMID:8985419

  9. Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection

    SciTech Connect

    DeLoach, J.R.; Andrews, K.; Sheffield, C.L.

    1988-04-01

    Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact (/sup 35/S)IL-2 was detectable in a number of tissues 3 days after injection.

  10. 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.

  11. Interleukin-2 in rheumatoid arthritis: production of and response to interleukin-2 in rheumatoid synovial fluid, synovial tissue and peripheral blood.

    PubMed Central

    Combe, B; Pope, R M; Fischbach, M; Darnell, B; Baron, S; Talal, N

    1985-01-01

    Several aspects of interleukin-2 (IL-2) generation and function were studied employing mononuclear cells from synovial fluid (SF), synovial tissue (ST) and peripheral blood (PB) of patients with rheumatoid arthritis (RA). Decreased PHA stimulated IL-2 production by lymphocytes from rheumatoid ST, SF (P less than 0.02), and PB (P less than 0.01) was observed when compared to normal blood and SF of patients with gout. The proliferative response of rheumatoid lymphocyte blasts exposed to exogenous IL-2 was also defective (P less than 0.05-0.001). This defect was greater in SF than in rheumatoid PB (P less than 0.05-0.001). In addition to the proliferative response, the effect of IL-2 on interferon-gamma (IFN-gamma) production was also examined. Rheumatoid lymphocytes from both PB and SF produced less IFN-gamma after overnight treatment with IL-2 than did normal PB lymphocytes. This decreased IFN-gamma induction was discordant with the excellent enhancement by IL-2 of natural killer activity. Removal of adherent cells in synovial fluid did not correct this deficit. Abnormalities in the biology of IL-2 and IFN-gamma suggest that impaired T cell function could contribute to the immunopathogenesis of RA. PMID:3921298

  12. High-dose continuous infusion plus pulse interleukin-2 and famotidine in melanoma.

    PubMed

    Quan, Walter; Ramirez, Maria; Taylor, W Chris; Vinogradov, Mikhail; Khan, Nawazish; Jackson, Shawn

    2004-12-01

    High-dose, continuous infusion interleukin-2 (IL-2) regimens generate greater Lymphokine Activated Killer cell (LAK) cytotoxicity in vitro and a higher rebound lymphocytosis in vivo than do bolus IL-2 regimens. Lymphocytes initially activated by continuous infusion IL-2 then subsequently pulsed with IL-2 have increased cytotoxicity against cancer cells. Famotidine may enhance the lysis of tumors by cytotoxic lymphocytes. Fourteen patients with melanoma were treated with famotidine 20 mg intravenously twice per day and continuous infusion IL-2 (18 MIU/sq m/24 hours) for 72 hours, followed by a 24-hour rest, then IL-2 18 MIU/sq m over 15-30 minutes for 1 dose (12 patients) or daily for 3 doses (2 patients). Most common toxicities were fever, nausea/emesis, hypophosphatemia, hypomagnesemia, and rigors. Nine partial responses (64% response rate; 95% Confidence Interval: 39%-84%) have been seen. Median survival has not been reached at greater than 10 months. Two patients responding to therapy showed an increase in detectable CD 56(+) cells in serial subcutaneous or lymph node biopsies, while 1 patient undergoing progression of disease had no such infiltrate. High-dose, 72-hour continuous infusion plus pulse interleukin-2 with famotidine has activity in melanoma. CD 56(+) cells may play a role in responding patients. PMID:15665626

  13. Continuous infusion interleukin-2 and antihistamines in melanoma: a retrospective review showing activity of this combination.

    PubMed

    Evangelista-Dean, Maria; Khan, Nawazish; Quan, Walter

    2004-12-01

    A recent randomized trial suggests that there may be an advantage in terms of survival with the combination of histamine and subcutaneous interleukin-2 (IL-2), compared to IL-2 alone. It has been postulated, then, that antihistamines may actually be antagonistic to IL-2 and, therefore, interfere with its antitumor activity. Because antihistamines such as cimetidine and ranitidine are commonly used as prophylaxis against gastrointestinal toxicity commonly seen with IL-2, and, because antihistamines may increase natural killer cell activity, it is reasonable to examine the response rate for this combination. An OVID Medline literature search between 1985 and 2003 was done. Continuous infusion (CIV) interleukin- 2 was used as the reference therapy because of the relatively constant IL-2 levels generated by this approach. Included studies were those in which either cimetidine, ranitidine, or famotidine were regularly scheduled and administered concurrently with IL-2, typically for gastrointestinal ulcer prophylaxis. Six (6) studies were identified. A total of 21 patients responded to therapy. Total response rate was 11%, with a 95% Confidence Interval: 7-17%. Four (4) complete responses were noted. Complete response rate was 2%, with a 95% Confidence Interval: 1-6%. These response rates are consistent with previously noted IL-2 response rates. In this retrospective review of CIV IL-2 and antihistamines, this combination appears to be active in melanoma. There appears to be no deleterious effect of routine antihistamine on the CIV IL-2 response rate. PMID:15665623

  14. l-Arginine modulates neonatal lymphocyte proliferation through an interleukin-2 independent pathway

    PubMed Central

    Yu, Hong-Ren; Kuo, Ho-Chang; Huang, Li-Tung; Chen, Chih-Cheng; Tain, You-Lin; Sheen, Jiunn-Ming; Tiao, Mao-Meng; Huang, Hsin-Chun; Yang, Kuender D; Ou, Chia-Yo; Hsu, Te-Yao

    2014-01-01

    In cases of arginine depletion, lymphocyte proliferation, cytokine production and CD3ζ chain expression are all diminished. In addition to myeloid suppressor cells, polymorphonuclear cells (PMN) also exert T-cell immune suppressive effects through arginase-induced l-arginine depletion, especially during pregnancy. In this study, we investigated how arginase/l-arginine modulates neonatal lymphocyte proliferation. Results showed that the neonatal plasma l-arginine level was lower than in adults (48·1 ± 11·3 versus 86·5 ± 14·6 μm; P = 0·003). Neonatal PMN had a greater abundance of arginase I protein than adult PMN. Both transcriptional regulation and post-transcriptional regulation were responsible for the higher arginase I expression of neonatal PMN. Exogenous l-arginine enhanced neonate lymphocyte proliferation but not that of adult cells. The RNA-binding protein HuR was important but was not the only modulation factor in l-arginine-regulated neonatal T-cell proliferation. l-Arginine-mediated neonatal lymphocyte proliferation could not be blocked by interleukin-2 receptor blocking antibodies. These results suggest that the altered arginase/l-arginine cascade may be one of the mechanisms that contribute to altered neonatal immune responses. Exogenous l-arginine could enhance neonate lymphocyte proliferation through an interleukin-2-independent pathway. PMID:24697328

  15. Activity of continuous infusion plus pulse interleukin-2 with famotidine in patients with metastatic kidney cancer or melanoma previously treated with interleukin-2.

    PubMed

    Quan, Walter D Y; Walker, Paul R; Quan, Francine M; Ramirez, Maria; Elsamaloty, Haitham M; Ghai, Vikas; Vinogradov, Mikhail; Liles, Darla K

    2006-10-01

    Lymphokine-activated killer (LAK) cells generated by high-dose continuous infusion interleukin-2 (IL-2) are able to nonspecifically lyse melanoma and kidney cancer cells. In vitro famotidine enhances cytotoxicity of LAK against tumor cells, possibly by increasing IL-2 uptake at the IL-2 receptor on lymphocytes. Outpatient IL-2 regimens typically have response rates of 15% or less, with most patients eventually experiencing progressive disease. Second-line therapy is, therefore, needed. We treated 11 patients (6 with metastatic melanoma; 5 having metastatic kidney cancer) who had previously experienced progressive disease on prior IL-2 regimens, with a combination of famotidine 20 mg intravenously (i.v.) twice per day and continuous-infusion IL-2 18 MIU/M2/24 hours x 72 hours, followed 24 hours later by a pulse IL-2 dose (18 MIU/M2 over 15 minutes). Cycles were repeated every 3 weeks. Patient characteristics were: 9 males, median age 63 years (range, 57-75), median Eastern Cooperative Oncology Group (ECOG) performance status: 1; most common metastatic sites: lungs, lymph nodes, and soft tissue/subcutaneous (s.c.); median number of cycles received: 4; most common toxicities were fever, nausea/emesis, hypophosphatemia, and hypomagnesemia. Five (5) patients (3 with melanoma, 2 with kidney cancer) have had partial responses. Two (2) patients with kidney cancer have been converted to complete responders with resection of residual disease, remaining without relapse at 5+ and 20+ months. Responding sites are lungs, lymph nodes, abdominal mass, and s.c. Median duration of response was 9.5 months. Median survival was 12 months. This combination has activity in patients with metastatic kidney cancer or melanoma who have received prior IL-2. PMID:17105418

  16. Constitutive secretion of soluble interleukin-2 receptor by human T cell lymphoma xenografted into SCID mice. Correlation of tumor volume with concentration of tumor-derived soluble interleukin-2 receptor in body fluids of the host mice.

    PubMed Central

    Wasik, M. A.; Sioutos, N.; Tuttle, M.; Butmarc, J. R.; Kaplan, W. D.; Kadin, M. E.

    1994-01-01

    Increased serum concentration of soluble alpha-chain receptor for interleukin-2 (sIL-2R) has been noted in patients with a variety of inflammatory conditions and lymphoid malignancies including T cell leukemia and lymphoma. Elevated sIL-2R serum levels seen in lymphoid malignancies appear to correlate with the clinical stage of disease. However, because sIL-2R is produced by normal activated lymphocytes, it has been uncertain whether serum sIL-2R in such conditions is derived from tumor cells or normal immune cells responding to the tumor. To address this question, we used a model of human (CD30+) anaplastic, large T cell lymphoma transplanted into immunodeficient SCID mice. Reverse transcription polymerase chain reaction of tumor RNA showed that the tumor, designated mJB6, contains mRNA for alpha-chain of human IL-2R. Furthermore, 15 to 25% of tumor cells stained with anti-human IL-2R alpha-chain mAb. Solid phase ELISA analysis of serum samples from mice bearing mJB6 lymphoma showed high concentrations of human sIL-2R. None of the control mice without lymphoma or with human nonlymphoid tumors (prostatic carcinoma, ovarian carcinoma, and glioblastoma multiforme) showed detectable human sIL-2R. The sIL-2R serum titers of mJB6-bearing mice correlated strongly with tumor volume (P < 0.0001). Tumors as small as 0.4 to 0.8 mm3 could be detected by this method. The sensitivity of sIL-2R ELISA exceeded at least 150 times the sensitivity of conventional radioisotopic tumor detection. Total resection of mJB6 tumors resulted in complete clearance of sIL-2R from the murine serum within 48 hours with a half-life of 6 hours. Accordingly, partial resection led to a significant decrease in sIL-2R followed by gradual increase with tumor regrowth. sIL-2R was also detected in the urine of mJB6-transplanted mice. As in serum, urine concentrations of sIL-2R were proportional to tumor mass (P < 0.02). Based on these findings we postulate that malignant cells are a major source of serum

  17. 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; Jörnvall, 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

  18. Colony formation and interleukin 2 production by leukaemic human T cells.

    PubMed Central

    Krajewski, A S; Dewar, A E; Seidelin, P H; Murray, R

    1983-01-01

    PHA-induced colony formation and interleukin 2 (IL-2) production were studied in four patients with T cell leukaemia (three cases OKT4+/T helper and one case OKT8+/T cytotoxic suppressor). Cases of T helper cell leukaemia showed colony formation that was comparable to normal purified blood T cells and was not dependent on the addition of conditioned medium, containing IL-2 activity, to cultures. In contrast the T suppressor cell leukaemia formed colonies only when cultures were supplemented with IL-2 containing medium. When IL-2 production by PHA stimulated cells was measured culture supernatants from the three T helper cell leukaemias all showed normal or high levels of activity, when compared to normal blood mononuclear cells, whereas the T suppressor cell leukaemia showed no activity. PMID:6604606

  19. Immunotherapy of murine sarcomas with interleukin 2. I. Local administration of human recombinant IL-2 preparations.

    PubMed

    Bubeník, J; Indrová, M; Toulcová, A

    1986-01-01

    The immunotherapeutic effect of human recombinant interleukin 2 was examined with a panel of MC-induced murine sarcomas carrying individual tumour-specific transplantation antigens. Repeated peritumoral injections of RIL-2 inhibited growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was resistant to the tumour-inhibitory effect of human recombinant IL-2 as well as to the tumour-inhibitory effect of murine and rat lymphoid IL-2 preparations. Since the IL-2-sensitive and IL-2-resistant sarcomas were induced with MC in mice of identical genotype and share most of their characteristics, they represent a useful model for investigation of structural target cell determinants and functional target cell properties responsible for the sensitivity of tumours to the immunotherapeutic effects of IL-2. PMID:3492396

  20. Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula.

    PubMed

    Young, L J; Cross, M L; Duckworth, J A; Flenady, S; Belov, K

    2012-01-01

    The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs. PMID:21683733

  1. Anti-colorectal cancer effect of interleukin-2 and interferon-β fusion gene driven by carcinoembryonic antigen promoter

    PubMed Central

    Wang, Yan; Wang, Mengchun; Li, Yan

    2016-01-01

    This study was designed to investigate the antitumor effects of combined interleukin-2/interferon-β-based gene therapy in colorectal cancer. Transfection of the fusion gene expression plasmid induced significant apoptosis of Lovo cells. Additionally, the fusion gene exhibited strong inhibitory activity against tumor growth and apoptosis when being injected into the nude mice implanted with human colon cancer cells. Furthermore, the tail-vein injection showed a more notable effect than direct injection into tumor. These results suggest that the combined interleukin-2/interferon-β-based gene therapy with the carcinoembryonic antigen promoter might be an effective antitumor strategy. PMID:27313471

  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

  5. Antagonistic effects of systemic interleukin 2 on immune Tcell-mediated graft-versus-leukemia reactivity.

    PubMed

    Schirrmacher, V; Müerköster, S; Umansky, V

    1998-11-01

    This study demonstrates that systemic interleukin 2 (IL-2) can decrease the homing of syngeneic immune T cells to the target organ of metastases and accelerate unwanted side effects of allogeneic immune T cells. As a tumor system, we used the well-characterized highly aggressive DBA/2 mouse leukemia ESb and its less aggressive adhesion variant, ESb-MP. Systemic IL-2 treatment was performed with recombinant human interleukin-2 (Proleukin), which was slowly released via an implanted osmotic pump or was modified with polyethylene glycol (PEG-IL-2) to achieve constant plasma levels. Allogeneic B10.D2 antitumor immune spleen cells (ISPL cells) exerted strong graft-versus-leukemia (GvL) reactivity after adoptive transfer into late-stage ESb-MP tumor-bearing DBA/2 mice. Mls(a) superantigen-reactive vbeta6 donor T cells were not eliminated or tolerized by in vivo priming with the tumor cells and were present in active proliferation in liver infiltrates. When exogenous PEG-IL-2 or Proleukin was applied in addition to ISPL cells in such mice, the strong GvL-mediated protective immunity was converted into a fatal graft-versus-host disease. IL-2 treatment alone had no toxic effect and caused a moderate protection effect in the absence of an effect on local tumor growth. Potentiation of GvH reactivity of B10.D2 ISPL by PEG-IL-2 was proven in non-tumor-bearing DBA/2 mice, in which graft-versus-host disease was characterized by: (a) heavy hepatic lymphocytic infiltration, (b) irreversible increase of serum glutamate-oxalacetate-transaminase and glutamate-pyruvate-transaminase levels, (c) weight loss, and (d) death. Antagonistic effects of systemic IL-2 on GvL were observed with syngeneic DBA/2 anti-ESb immune peritoneal effector cells (PECs). There was a detrimental effect of systemic IL-2 on liver target organ infiltration by immune T cells causing, at day 6 after transfer, a drop from 20-30 CD4 or CD8 T cells per liver lobule in the PEC group to <5 in the PEC plus IL-2 group

  6. Pentoxifylline inhibits interleukin-2-induced toxicity in C57BL/6 mice but preserves antitumor efficacy.

    PubMed Central

    Edwards, M J; Heniford, B T; Klar, E A; Doak, K W; Miller, F N

    1992-01-01

    Interleukin 2 (IL-2) mediates the regression of metastatic cancer but clinical use has been limited due to associated toxicities. Tumor necrosis factor (TNF) is an important mediator of IL-2 toxicity and may have a limited role in IL-2 antitumor efficacy. Because pentoxifylline (PTXF) inhibits TNF production, we hypothesized that PTXF would ameliorate IL-2 toxicity without compromising antitumor efficacy. Four groups of female C57BL/6 mice with pulmonary metastases from a 3-methylcholanthrene-induced fibrosarcoma (MCA-105) and four groups of nontumored mice were treated every 6 h for 4 d by intraperitoneal injections of either IL-2 alone, IL-2 and PTXF, PTXF alone, or equal volumes of saline. Upon completion of therapy, we found that PTXF suppressed many of the IL-2-induced effects including TNF production, lymphocytic infiltration of multiple organs, multiple organ edema, hepatic dysfunction, leukopenia, and thrombocytopenia. Tumor response was determined 21 d after cessation of therapy by quantitating the number and surface area of pulmonary metastases. PTXF preserved antitumor efficacy while reducing the morbidity and mortality caused by IL-2 treatment. These data strongly support the use of PTXF in extending the therapeutic index of IL-2 in the treatment of cancer. Images PMID:1644928

  7. High-dose continuous infusion plus pulse interleukin-2 and famotidine in metastatic kidney cancer.

    PubMed

    Quan, Walter; Ramirez, Maria; Taylor, Chris; Vinogradov, Mikhail; Quan, Francine; Khan, Nawazish

    2005-02-01

    High-dose continuous infusion interleukin-2 (IL-2) regimens generate a higher degree of lymphokine activated killer cell (LAK) cytotoxicity when tested against tumor cells in vitro and a higher rebound lymphocytosis in vivo than do bolus IL-2 regimens. Lymphocytes initially activated by continuous infusion IL-2 have increased cytotoxicity against cancer cells when they are subsequently pulsed with additional IL-2. Famotidine may enhance LAK cytolytic ability. Six patients with kidney cancer have been treated with a combination of famotidine 20 mg intravenous bid and continuous infusion IL-2 (18 MIU/sq m/24 hours) for 72 hours, followed by a 24-hour rest, then IL-2 18 MIU/sq m over 15-30 minutes. The most common metastatic sites were the lung, lymph node, and bone. Median number of cycles received = 5 (range, 3-8). The most common toxicities were fever, rigors, nausea/emesis, hypophosphatemia, hypotension, elevated creatinine, and metabolic acidosis. There were no treatment-related deaths, and no patients required intensive care admission. Two partial responses (33% response rate) have been seen. Median survival has not been reached at greater than 8 months. The combination of high-dose continuous infusion plus pulse IL-2 and famotidine is active in metastatic kidney cancer. An accrual of additional patients is needed to better assess the response rate. PMID:15778577

  8. Activity of continuous infusion + pulse interleukin-2 with famotidine in metastatic melanoma.

    PubMed

    Quan, Walter D Y; Quan, Francine M

    2009-02-01

    High-dose interleukin-2 (IL-2), given via continuous intravenous (i.v.) infusion, induces lymphokine-activated killer (LAK) cell cytotoxicity against tumor cells. These LAKs exhibit enhanced cytotoxicity against tumor cells in vitro when they are subsequently pulsed with additional IL-2. Famotidine may increase LAK cytotoxicity against neoplastic cells by allowing for greater IL-2 uptake at the IL-2 receptor on lymphocytes. Twenty-three (23) patients received famotidine 20 mg i.v. twice per day and continuous-infusion IL-2 (18 MIU/m(2)/24 hours) for 72 hours, followed by a 24-hour rest, then 1-3 daily-pulse IL-2 doses of 18 MIU/m(2) over 15-30 minutes preceded by famotidine 20 mg i.v. Cycles were repeated every 3 weeks. The most common metastatic sites were lung, lymph node, and subcutaneous/soft tissue. The most common toxicities were fever, rigor, nausea/emesis, hypophosphatemia, hypotension, elevated creatinine, and pulmonary edema. There were no treatment-related deaths. One (1) complete (4%) and 9 partial responses (39%) were seen (43% total response rate; 95% confidence interval: 22%-65%). Median survival for all patients is 13 months. The combination of famotidine and high-dose continuous infusion + pulse IL-2 is active in metastatic melanoma. PMID:19243244

  9. High-dose intensity pulse interleukin-2 with famotidine has activity in metastatic melanoma.

    PubMed

    Quan, Walter D Y; Walker, Paul R; Picton, Maria; Quan, Francine M; King, Linda A; Tyre, Charley; Liles, Darla K

    2008-10-01

    Daily short intravenous (i.v.) infusions (pulses) of interleukin-2 (IL-2) have been developed to decrease toxicity while maintaining anticancer activity of this agent against melanoma. Such IL-2 schedules have previously been shown to promote lymphokine-activated killer (LAK) cell activity. Famotidine may increase LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. We treated 16 patients with metastatic melanoma using pulse IL-2 18 (15 patients) or 9 million IU/M2 (1 patient) i.v. over 15-30 minutes preceded by famotidine 20 mg i.v. daily for 5 days on an oncology inpatient unit. Cycles were repeated every 3 weeks until disease progression. Patient characteristics were as follows: 11 males, median age, 66, median ECOG performance status, 1; common metastatic sites: lymph nodes, lungs, subcutaneous, liver, and bone. Median number of cycles received was 3. Overall, 93% of planned doses were delivered. Most common toxicities were hypomagnesemia, fever, rigors, hypophosphatemia, and nausea/emesis. Three (3) patients had partial responses (19% response rate; 95% confidence interval: 6%-44%). A fourth patient, after resection of residual disease, remains a surgical complete responder at > 12 months. Responses occurred in lung, liver, lymph nodes, bone, and subcutaneous sites. Median response duration was 7 months. Pulse IL-2 with famotidine has activity in melanoma. PMID:18999936

  10. High-dose intensity pulse interleukin-2 with famotidine in metastatic kidney cancer.

    PubMed

    Quan, Walter D Y; Quan, Francine M

    2009-04-01

    Lymphokine-activated killer cell (LAK) activity against tumor cell lines may be induced by intravenous (i.v.) interleukin-2 (IL-2). Daily short infusions (pulses) have been developed to decrease toxicity while maintaining the anticancer activity of this agent against kidney cancer. The anthihistamine, famotidine, may increase IL-2 uptake by the IL-2 receptor on lymphocytes. We have treated 12 patients with metastatic kidney cancer, using pulse IL-2 (18 million IU/M(2) i.v.) over 15-30 minutes, preceded by famotidine (20 mg I.V. daily for 5 days) on an oncology inpatient unit. Cycles were repeated every 3 weeks until disease progression. Patient characteristics were as follows: 9 males with a median age of 66 years (range, 48-74), and median Eastern Cooperative Oncology Group performance status of 1; common metastatic sites included in the lungs 9 and lymph nodes 3. Median number of cycles received was 2 (range, 1-5). The most common toxicities were fever, rigors, and hypomagnesemia. Two (2) patients had partial responses (17% response rate). Responses occurred in the liver (11.5 months) and lung, pleura, and lymph nodes (3 months). Pulse IL-2 with famotidine shows activity in kidney cancer. PMID:19409039

  11. Activity of outpatient intravenous interleukin-2 and famotidine in metastatic clear cell kidney cancer.

    PubMed

    Quan, Walter D Y; Quan, Francine Marie

    2014-03-01

    Outpatient daily intravenous infusions of interleukin-2 (IL-2) have been developed to maintain anticancer activity and decrease toxicity of this agent against kidney cancer. Lymphokine activated killer cell (LAK) numbers are increased with these IL-2 schedules. Famotidine may enhance the LAK activity by increasing IL-2 internalization by the IL-2 receptor on lymphocytes. Fifteen patients with metastatic clear cell kidney cancer received IL-2 18 million IU/M² intravenously over 15-30 minutes preceded by famotidine 20 mg IV daily for 3 days for 6 consecutive weeks as outpatients. Cycles were repeated every 8 weeks. Patient characteristics were seven males/eight females, median age 59 (range: 28-70), median Eastern Cooperative Oncology Group (ECOG) performance status-1; common metastatic sites were lungs (14), lymph nodes (9), liver (4), bone (4), and pancreas (4). Prior systemic therapies were oral tyrosine kinase inhibitor (8), IL-2 (6), and mTor inhibitor (2). Most common toxicities were rigors, arthralgia/myalgia, nausea/emesis, fever, and hypotension. All episodes of hypotension were reversible with intravenous fluid. No patients required hospitalization due to toxicity. One complete response (7%) and four partial responses (26%) were seen (total response rate=33%; 95% confidence interval: 15%-59%). Responses occurred in the lungs, liver, lymph nodes, and bone. Outpatient intravenous IL-2 with famotidine has activity in metastatic clear cell kidney cancer. PMID:24251758

  12. 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

  13. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.

    PubMed Central

    Wakao, H; Harada, N; Kitamura, T; Mui, A L; Miyajima, A

    1995-01-01

    Signal transducers and activators of transcription (STAT) proteins play an important role in cytokine signal transduction in conjunction with Janus kinases (JAKs). MGF/STAT5 is known as prolactin regulated STAT. Here we demonstrate that interleukin 2 (IL-2) as well as erythropoietin (EPO) stimulate STAT5 and induce tyrosine phosphorylation of STAT5. These IL-2- and EPO-induced STATs have an identical DNA binding specificity and immunoreactivity. We also show that IL-4 induces a DNA binding factor which possesses similar, but distinct, DNA binding specificity from that of STAT5 and is immunologically different from STAT5. Analysis of two EPO receptor (EPOR) transfected CTLL-2 cell lines discloses that IL-2 activates JAK1 and JAK3 as well as STAT5, while EPO stimulates STAT5 and JAK2 in EPO-responsive CTLL-2 cells (ERT/E2). On the contrary, EPO activates neither JAK2 nor STAT5 in other cell lines that failed to respond to EPO (ERT cells). EPOR and JAK2 associate with each other regardless of EPO presence in ERT/E2 cells, however, such an interaction is not present in ERT cells. Thus, EPOR and JAK2 association seems to be important for EPO responsiveness in CTLL-2 cells. Images PMID:7781605

  14. Interleukin 2 receptor in patients with localized and systemic parasitic diseases.

    PubMed Central

    Josimovic-Alasevic, O; Feldmeier, H; Zwingenberger, K; Harms, G; Hahn, H; Shrisuphanunt, M; Diamantstein, T

    1988-01-01

    An enzyme-linked immunosorbent assay was used to quantify soluble interleukin 2 receptor (IL-2R) in the serum of patients with helminthic and protozoal infections. The results demonstrated that levels of IL-2R were normal in patients with helminthic infections limited to the intestinal tract (ascariasis, trichuriasis), but significantly elevated in patients with systemic or long-lasting infections (strongyloidiasis, schistosomiasis, fascioliasis, opisthorchiasis). In patients infected with Schistosoma mansoni levels of IL-2R were higher in those with the hepatosplenic than in those with the intestinal form of the disease. Patients with malaria also showed increased serum levels of IL-2R, irrespective whether the infection was caused by Plasmodium falciparum or P. vivax. No difference was observed between patients with acute or history of malaria. The highest levels of IL-2R were observed in patients with visceral leishmaniasis. Interestingly, in these patients the concentration of IL-2R correlated to specific antibody titre. The results are discussed in the context of preferential activation of T lymphocytes, B lymphocytes and/or macrophages during the course of the different parasitic infections investigated. PMID:3136958

  15. 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.

  16. Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment*

    PubMed Central

    de Oca B., Pavel Montes; Malardé, Valerie; Proust, Richard; Dautry-Varsat, Alice; Gesbert, Franck

    2010-01-01

    Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Rα, -β, and -γc). IL-2Rβ is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rβ is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37βic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rβ decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37βic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rβ mimicking 37βic increases their proliferation. These data indicate that IL-2Rβ is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation. PMID:20495002

  17. Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.

    PubMed Central

    Kelley, V E; Bacha, P; Pankewycz, O; Nichols, J C; Murphy, J R; Strom, T B

    1988-01-01

    De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response. Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH). A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli [Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B. & Murphy, J.R. (1987) Protein Eng. 1, 493-498]. We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH. The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent. Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells. Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes. DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid. PMID:3131768

  18. Thyroid dysfunction in 281 patients with metastatic melanoma or renal carcinoma treated with interleukin-2 alone.

    PubMed

    Krouse, R S; Royal, R E; Heywood, G; Weintraub, B D; White, D E; Steinberg, S M; Rosenberg, S A; Schwartzentruber, D J

    1995-11-01

    The purpose of this prospective study was to determine the incidence of thyroid dysfunction in cancer patients receiving immunotherapy with interleukin-2 (IL-2) alone, and to assess the relationship of hypothyroidism to clinical response. A cohort of 281 consecutive patients with metastatic melanoma or renal carcinoma were treated with IL-2 alone from July 1, 1989 until June 30, 1993. The majority (n = 216) received high-dose IL-2 and the remainder (n = 65) received low-dose therapy. Thyroid function was measured before, during, and after immunotherapy. Forty-one percent of initially euthyroid patients developed thyroid dysfunction after starting high-dose IL-2-alone therapy. The most common abnormality was hypothyroidism, occurring in 35% of patients, although moderate or severe hypothyroidism requiring thyroid hormone replacement occurred in 9% of patients. Hypothyroidism was related to duration of IL-2 therapy and was not associated with clinical response. Hyperthyroidism developed in 7% of previously euthyroid patients receiving high-dose IL-2. Overall, the incidence of thyroid dysfunction was similar in the high- and low-dose IL-2 regimens. In conclusion, thyroid dysfunction is a common sequela of IL-2 therapy. Thyroid function should be measured routinely in cancer patients receiving IL-2-based treatment. It is recommended that thyroid hormone replacement be given to patients with moderate or severe hypothyroidism. PMID:8680655

  19. Systemic induction of cells mediating antibody-dependent cellular cytotoxicity following administration of interleukin 2.

    PubMed

    Eisenthal, A; Rosenberg, S A

    1989-12-15

    We have previously demonstrated that incubation of murine cells in vitro in interleukin 2 (IL-2) induced antibody-dependent cellular cytotoxicity (ADCC) and that these cells were derived from the NK/LAK, FcR+ cell population. In the present study we show that in vivo administration of IL-2 to mice induces cells which exhibit ADCC activity in the peritoneal cavity, liver, lungs, and to a lesser degree in the bone marrow, spleen, mesenteric lymph nodes, and thymus. A gradual increase in ADCC activity and the number of Fc-receptor-positive cells was seen 1 to 3 days after starting IL-2 treatment. The cells mediating ADCC are closely related to LAK cells since they expressed Thy1.2 antigens and are derived from asialo GM1-positive, Lyt2/L3T4-negative, radiosensitive cells. These results demonstrate that IL-2 can systemically induce cells with ADCC activity and that this ability may be useful in the establishment of therapeutic models against disseminated cancer when combined with specific antitumor monoclonal antibodies. PMID:2573425

  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-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ 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-γ and NF-AT. PMID:21435270

  1. Interleukin 2 Gene Polymorphisms Are Associated with Non-Hodgkin Lymphoma

    PubMed Central

    Song, Haihan; Chen, Lei; Cha, Zhanshan

    2012-01-01

    Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy worldwide. Interleukin-2 (IL-2) plays a key role in the proliferation of T cells and natural killer cells. It has been reported that polymorphisms in the IL-2 gene are associated with various cancers. The aim of this study was to examine the effect of polymorphisms in the IL-2 gene on the development of NHL in the Chinese population. IL-2-330T/G and +114T/G polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism in 438 NHL cases and 482 age-matched healthy controls. Data were analyzed using the Chi-square test. Results showed that individuals with −330TG genotype or −330GG genotype had significantly increased susceptibility to NHL (Odds ratio [OR]=1.40, 95% confidence interval [CI]: 1.05–1.85, p=0.020 and OR=2.04, 95%CI: 1.28–3.24, p=0.002). Meanwhile, the +114T/G polymorphism did not show any correlation with NHL. When analyzing the haplotypes of these two polymorphisms, the prevalence of −330G/+114T haplotype was significantly higher in NHL cases than in controls (OR=1.45, 95%CI: 1.12–1.88, p=0.005). These data indicate that IL-2 gene polymorphisms may be new risk factors for NHL. PMID:22472080

  2. Soluble interleukin-2 receptor: elevated levels in serum and synovial fluid of patients with rheumatoid arthritis.

    PubMed

    Carpenter, A B; Eisenbeis, C H; Carrabis, S; Brown, M C; Ip, S H

    1990-01-01

    Soluble interleukin-2 receptor (sIL-2R) levels were quantitated in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and degenerative joint disease (DJD). A sandwich immunoassay, employing two monoclonal antibodies against distinct epitopes on the IL-2R, was utilized for measurement. We found a striking elevation of sIL-2R in RA SF as compared with DJD SF (RA, 1319 +/- 135; DJD, 416 +/- 59; p less than 0.001). RA serum sIL-2R levels were also significantly elevated over DJD levels. There was no interaction between rheumatoid factor (RF) and sIL-2R. RA patients with elevated sIL-2R levels had significantly longer disease duration, higher c-reactive protein (CRP) levels in serum and SF, and higher RF levels in serum and SF. The groups were similar in regard to other laboratory variables. The presence of elevated levels of sIL-2R in RA serum and SF confirms the presence of a heightened immune reactivity and in vivo activation of lymphocytes in RA. PMID:2313471

  3. Annexin A6 regulates interleukin-2-mediated T-cell proliferation.

    PubMed

    Cornely, Rhea; Pollock, Abigail H; Rentero, Carles; Norris, Sarah E; Alvarez-Guaita, Anna; Grewal, Thomas; Mitchell, Todd; Enrich, Carlos; Moss, Stephen E; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-07-01

    Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4(+) T cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment. PMID:26853809

  4. Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

    SciTech Connect

    Zu, Youli; Kohno, Michiaki; Namba, Yuziro ); Kohno, Michiaki ); Kubota, Ichiro ); Nishida, Eisuke )

    1990-01-30

    The authors have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of ({sup 32}P)P{sub i}-labeled and ({sup 3}H)leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fraction studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.

  5. High-dose interleukin 2-induced myocarditis: can myocardial damage reversibility be assessed by cardiac MRI?

    PubMed

    Chow, Shien; Cove-Smith, Laura; Schmitt, Matthias; Hawkins, Robert

    2014-06-01

    High-dose interleukin 2 (HD-IL2) is one of the therapeutic options for patients with metastatic renal cell carcinoma. In well-selected patients with favorable clinical and pathologic features, it offers impressive response and potential long-term remission. It also has a place for treatment for metastatic malignant melanoma and in adoptive cell therapy. However, it is known for its intensive course and toxicities. Myocarditis is one of the known complications of this treatment and can pose a diagnostic challenge to treating oncologists because of its nonspecific and similar presentation to acute coronary syndrome (ACS). We report 3 short cases of HD-IL2-related myocarditis, which were either missed or misdiagnosed as ACS using conventional assessment but subsequently accurately diagnosed by cardiac magnetic resonant imaging (CMR). We discussed the clinical presentation of these cases and demonstrated the diagnostic advantage of CMR compared with standard investigations including its superior capability to assess myocardial reversibility, which has important short-term and long-term implications. The use of CMR also avoided unnecessary invasive intervention such as coronary angiogram in all 3 patients. These example cases call for effort to conduct prospective research to assess and confirm the utility of CMR, thus informing a more effective management pathway for immune-related myocarditis in HD-IL2 and other cancer immunotherapy. PMID:24810642

  6. 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.

  7. A novel and simple type of liposome carrier for recombinant interleukin-2.

    PubMed

    Kanaoka, E; Takahashi, K; Yoshikawa, T; Jizomoto, H; Nishihara, Y; Hirano, K

    2001-03-01

    The strong interaction between recombinant interleukin-2 (IL-2) and liposome was characterized and its possible application to drug-delivery control considered. The liposomes were prepared with egg phosphatidylcholine, distearoyl-phosphatidylglycerol (DSPG), dipalmitoyl-phosphatidylcholine, dipalmitoyl-phosphatidylglycerol or distearoyl-phosphatidylcholine (DSPC). Small and hydrophobic liposomes were selected, which were composed of saturated and long-fatty-acid-chain phospholipids. When the composition and the mixture ratio of IL-2 and the liposomewere optimized, morethan 95% ofthe lyophilized IL-2 (Imunace, 350000 JRU) was adsorbed consistently onto the DSPC-DSPG liposome (molar ratio, 10:1; 25 micromol mL(-1); 30 nm in size). Merely mixing IL-2 lyophilized with liposome suspension is convenient pharmaceutically. After intravenous administration to mice, liposomal IL-2 was eliminated half as slowly from the systemic circulation as free IL-2, with more than 13 and 18 times more IL-2 being delivered to the liver and spleen, respectively. After subcutaneous administration of liposomal IL-2 to mice, the mean residence time of IL-2 in the systemic circulation was 8 times that of free IL-2. These results show that IL-2 consistently adsorbs onto the surface of liposomes after optimization of its composition and mixing ratio. Intravenous and subcutaneous administration to mice demonstrates the gradual release of IL-2. Further trials are warranted using these liposomes. PMID:11291744

  8. Interleukin-2 and interferon-alpha-2a outpatient therapy for metastatic renal cell carcinoma.

    PubMed

    Lipton, A; Harvey, H; Givant, E; Hopper, K; Lawler, J; Matthews, Y; Hirsh, M; Zeffren, J

    1993-02-01

    The combination of interleukin-2 (IL-2) and interferon-alpha-2a (IFN-alpha-2a) has synergistic bioactivity in numerous preclinical model systems. Thirty-nine patients with metastatic renal cell cancer were treated with continuous intravenous infusion IL-2 for 4-5 days plus intramuscular IFN-alpha-2a 2-3 days a week for 4 consecutive weeks. A 2- to 4-week rest period was permitted after each 4 weeks of treatment. Thirty-one of the 39 patients were assessable for response determination. Response rate (six complete+seven partial remissions) was 33.3% for all patients, or 41.9% when the analysis was restricted to the 31 evaluable patients. Three patients were unable to tolerate treatment due to anorexia, weight loss, and severe fatigue. This therapy was relatively well tolerated in the outpatient setting in the other patients despite fever, chills, fatigue, anorexia, and weight loss. There was no correlation of response with site of metastases or bulk of disease. PMID:8318497

  9. Clinical and immunological effects of single bolus administration of recombinant interleukin-2 in cattle.

    PubMed Central

    Campos, M; Hughes, H P; Godson, D L; Sordillo, L M; Rossi-Campos, A; Babiuk, L A

    1992-01-01

    Recombinant bovine interleukin-2 (rBoIL-2) was administered as a single intramuscular bolus to healthy calves to determine the minimal dose capable of exerting a biological response. Doses ranging from 2.5 to 0.05 micrograms rBoIL-2/kg did not induce pyrexia, diarrhea, or depression, nor did they alter any blood chemistry or hematological parameters commonly associated with IL-2 toxicity. Moreover, the only significant immunological change observed was a reduction in the number of peripheral blood lymphocytes identified with the monoclonal antibodies B7A, BAQ4A (WC1+ cells), CACTB6A (WC2+ cells) and DH59B (monocytes). The decrease in cells associated with these markers did not influence non-MHC restricted cytotoxicity or in vitro lymphocyte proliferative responses to mitogens and IL-2. The treatments had no effect on delayed type hypersensitivity responses to phytohemagglutinin. These results indicate that IL-2 may be involved in the regulation of trafficking patterns of a unique subpopulation of lymphocytes in cattle. PMID:1586889

  10. Increased production of interleukin-2 and IL-2 receptor in primary IgA nephropathy.

    PubMed

    Schena, F P; Mastrolitti, G; Jirillo, E; Munno, I; Pellegrino, N; Fracasso, A R; Aventaggiato, L

    1989-03-01

    The production of interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBMC) in 13 patients with IgA nephropathy (IgAN) and 9 patients with chronic glomerulonephritis was investigated. Moreover, the distribution of IL-2 receptor (IL-2R) expression was studied in the purified T cell population versus the non-T cell population of IgAN patients. The results show a spontaneous significant production of IL-2 in cultures of PBMC from patients with IgAN (P less than 0.025) that increased after PHA stimulation. IgAN patients also had a significantly higher expression of IL-2R on the surface of PBMC than did patients with chronic glomerulonephritis (P less than 0.05). IL-2R was usually detected on unstimulated purified T cells that expressed the activation DR antigen. Moreover, a high number of DR helper T cells was associated to a reduced number of suppressor T cells (OKT8+M1+). These findings suggest that the increased production of IL-2 in patients with IgAN may be responsible for the increased activity of helper T cells. The high number of IL-2R expressed by freshly separated PBMC implies an in vivo continuous stimulation of these cells, and this finding is in agreement with the demonstrated spontaneous hyperproduction of IL-2. Moreover, the low number of suppressor T cells may contribute to the overactivity of helper T cells bearing IL-2R in IgAN patients. PMID:2785227

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

    SciTech Connect

    Cervia, Davide; 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.

  12. Effect of anti-interleukin 2 monoclonal antibody treatment on the survival of rat cardiac allograft

    SciTech Connect

    Sakagami, K.; Ohsaki, T.; Ohnishi, T.; Saito, S.; Matsuoka, J.; Orita, K.

    1989-03-01

    The effect of anti-interleukin 2 monoclonal antibody (anti-IL2 MoAb) and the accumulation of intravenously administered /sup 125/I-labeled anti-IL2 MoAb were examined in heterotopic rat cardiac allografts. Mouse anti-human recombinant IL2 MoAb was obtained by the hybridoma technique. The anti-IL2 MoAb, termed 8H-10, was an IgG2a which inhibited IL2-driven (/sup 3/H)TdR incorporation in cytolytic T lymphocyte line cells at a dilution of 2(6). 8H-10 was injected iv at a dose of 200 micrograms/day for 8 consecutive days, beginning on the day of transplantation. Hearts from F344 rats (RT11v1) were transplanted into ACI recipient rats (RT1av1). The mean survival time was 7.6 +/- 0.8 days in untreated controls, 9.0 +/- 1.2 days in additional controls treated with mouse anti-sheep red blood cell monoclonal antibody, and 25.3 +/- 18.4 days in the anti-IL2 MoAb (8H-10)-treated group (P less than 0.05). Furthermore, the accumulation of intravenously administered 125I-labeled anti-IL2 MoAb (8H-10) was specifically seen in the grafted heart. In conclusion, these results suggest that IL2 may play an important role in allograft rejection and that anti-IL2 MoAb may serve as a useful immunosuppressive agent in clinical transplantation.

  13. Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.

    PubMed Central

    Burton, J; Goldman, C K; Rao, P; Moos, M; Waldmann, T A

    1990-01-01

    Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction. Images PMID:1976256

  14. Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment.

    PubMed

    Lee, J; Fenton, B M; Koch, C J; Frelinger, J G; Lord, E M

    1998-04-01

    Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy. Here, we examine the hypoxic state of EMT6 mouse mammary tumors and the location of host cells within the different areas of the tumors to determine whether such microenvironmental conditions might also affect their ability to be recognized by the immune system. Hypoxia within tumors was quantified by flow cytometry and visualized by immunohistochemistry using a monoclonal antibody (ELK3-51) against cellular adducts of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5), a nitroimidazole compound that binds selectively to hypoxic cells. Thy-1+ cells, quantified using a monoclonal antibody, were found only in the well-oxygenated areas. The location of these Thy-1+ cells was also examined in EMT6 tumors that had been transfected with the gene for interleukin-2 (IL-2) because these tumors contain greatly increased numbers of host cells. Surprisingly, we found that IL-2-transfected tumors had significantly decreased hypoxia compared to parental tumors. Furthermore, using the fluorescent dye Hoechst 33342, an in vivo marker of perfused vessels, combined with immunochemical staining of PECAM-1 (CD31) as a marker of tumor vasculature, we found increased vascularization in the IL-2-transfected tumors. Thus, expression of IL-2 at the site of tumor growth may enhance tumor immunity not only by inducing the generation of tumor-reactive CTLs but also by allowing increased infiltration of activated T cells into the tumors. PMID:9537251

  15. 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

  16. Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.

    PubMed Central

    Jacobson, E L; Pilaro, F; Smith, K A

    1996-01-01

    When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions. Images Fig. 1 Fig. 2 PMID:8816813

  17. 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

  18. 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-γ 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.

  19. HLA association with response and toxicity in melanoma patients treated with interleukin 2-based immunotherapy.

    PubMed

    Marincola, F M; Venzon, D; White, D; Rubin, J T; Lotze, M T; Simonis, T B; Balkissoon, J; Rosenberg, S A; Parkinson, D R

    1992-12-01

    Peripheral blood lymphocytes from 146 patients with metastatic melanoma undergoing interleukin 2 (IL-2)-based immunotherapy were characterized for HLA A, B, Cw, DR, DQw, and DRw specificities. Patients had been enrolled into sequential treatment protocols with either IL-2 alone (28) or in combination with tumor-infiltrating lymphocytes (TILs) (86), alpha-interferon (26), lymphokine-activated killer cells (16), radiation therapy (7), cyclophosphamide (3), tumor necrosis factor (1), and interleukin 4 (1) for a total of 168 courses of therapy. HLA phenotype was then correlated with response rate and toxicity to IL-2. We noted: (a) a significant difference in the frequency of A11 (20.5% versus 10.2%; P < 0.05) allele between melanoma patients and the North American Caucasian population; (b) a significantly higher frequency of A11 phenotype among responders (40.5%) than in the melanoma patient population (20.5%; P < 0.01), which was even more obvious among patients responding to TIL therapy (47.4% versus 22.1%; P < 0.05); within TIL patients, responders also had an increased frequency of A19 (42.1% versus 25.6%; P < 0.05); (c) a correlation between the number of TILs received and response rate (P < 0.005); and (d) an association between DR4 haplotype and decreased tolerance to IL-2 among the patients receiving TILs (P = 0.01). These results suggest that, in melanoma patients, some HLA Class I specificities may predict for a greater likelihood of response to IL-2-based therapy, while HLA Class II phenotype correlates with tolerance to the combination of TIL and IL-2 therapy. PMID:1423301

  20. Association between two interleukin-2 gene polymorphisms and cancer susceptibility: a meta-analysis

    PubMed Central

    Zhang, Meng; Tan, Xiuxiu; Huang, Junjie; Xie, Lijuan; Wang, Hao; Shi, Jizhou; Lu, Wei; Lv, Zhaojie; Mei, Hongbing; Liang, Chaozhao

    2016-01-01

    Background Several epidemiological studies have illustrated that polymorphisms in interleukin-2 (IL-2) were associated with diverse cancer types. However, recently published statistics were inconsistent and inconclusive. Therefore, the current meta-analysis was performed to elaborate the effects of IL-2 polymorphisms (rs2069762 and rs2069763) on cancer susceptibility. Material and methods A total of 5,601 cancer cases and 7,809 controls from 21 published case–control studies were enrolled in our meta-analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between IL-2 polymorphisms and cancer susceptibility. Results Our study demonstrated an increased susceptibility to cancer in rs2069762 (G vs T: OR =1.268, 95% CI =1.113–1.445; GG vs TT: OR =1.801, 95% CI =1.289–2.516; GT vs TT: OR =1.250, 95% CI =1.061–1.473; GG + GT vs TT: OR =1.329, 95% CI =1.118–1.579; GG vs GT + TT: OR =1.536, 95% CI =1.162–2.030). In the subgroup analysis, increased susceptibility to cancer was identified in the hospital-based group and PHWE<0.05 (P-value of the Hardy–Weinberg equilibrium [HWE]) group. In addition, a positive association with cancer susceptibility was observed among both Chinese and non-Chinese. However, no relationship was detected between the rs2069763 polymorphism of IL-2 and cancer susceptibility. Conclusion To conclude, rs2069762 polymorphism of IL-2 contributed to an increased susceptibility to cancer, whereas no association was identified between rs2069763 polymorphism and cancer susceptibility. Further detailed studies are warranted to confirm our findings. PMID:27143914

  1. Interleukin-2 in relation to T cell subpopulations in rheumatic heart disease.

    PubMed Central

    Zedan, M M; el-Shennawy, F A; Abou-Bakr, H M; al-Basousy, A M

    1992-01-01

    Interleukin-2 (IL-2) and T cell subpopulations were evaluated in children with rheumatic heart disease (RHD). Three groups were included: 13 patients with active RHD, 12 with non-active RHD, and 14 control children. Serum IL-2 and T cell subpopulations were measured by radioimmunoassay and monoclonal antibodies respectively. Patients with active RHD showed a significant increase in IL-2 concentrations and helper:suppressor (H:S) ratio compared with controls with a mean (SEM) IL-2 of 3.48 (0.62) v 1.26 (0.16) U/ml and H:S ratio 2.31 (0.14) v 1.66 (0.04). There was a significant decrease in T suppressor (CD8+) and pan T (CD3+) cells compared with controls with a mean (SEM) for CD8+ of 23.75 (1.19) v 32.23 (0.56)% and CD3+ of 79.55 (0.94) v 85.00 (0.11)%. Patients with non-active RHD showed a significant decrease only in the CD3+ cells (78.20 (0.20)%) when compared with controls. A deficiency of CD3+ cells is a constant finding in patients with RHD, whether the disease is active or not. There was a significant increase in IL-2 concentration with a significant decrease in CD8+ cells in patients with active RHD in comparison with the non-active group (mean (SEM) IL-2 of 3.48 (0.62) v 1.85 (0.24) U/ml and CD8+ of 23.75 (1.19) v 28.83 (1.91)%). Thus an increase in IL-2 and a decrease in CD8+ cells may be related to rheumatic activity. T helper (CD4+) cells did not differ significantly between groups. PMID:1471890

  2. Clinical significance of serum soluble interleukin-2 receptor in chronic myeloproliferative disorders.

    PubMed

    Kawatani, T; Endo, A; Tajima, F; Ooi, S; Kawasaki, H

    1997-02-01

    Serum soluble interleukin-2 receptor (sIL-2R) levels were determined in patients with chronic myeloproliferative disorders (CMPD): 18 with chronic myelogenous leukemia in chronic phase (CML in CP), seven with CML in accelerated phase (AP) or blastic crisis (BC), six with polycythemia vera (PV), eight with essential thrombocythemia (ET), one with primary myelofibrosis (PMF), and 50 controls. The mean (+/-S.E.M.) levels were higher in CMPD than in controls (CML in AP or BC, 2693 +/- 694 U/ml, P < 0.0001; CML in CP, 792 +/- 63 U/ml, P < 0.0001; PV 553 +/- 89 U/ml, P < 0.05; ET, 449 +/- 56 U/ml; PMF, 628 U/ml vs. controls, 395 +/- 25 U/ml). Patients with CML in CP had significantly higher serum sIL-2R levels than patients with ET (P < 0.005), and levels were markedly elevated in AP and BC (P < 0.001). Serum sIL-2R levels were positively correlated with WBC count and lactic dehydrogenase in CMPD, and in CML in CP. Serum sIL-2R levels in CMPD were negatively correlated with RBC and platelet counts. Serum sIL-2R levels were significantly lower in patients with CML in CP who showed a cytogenetic response after interferon (IFN) therapy than in those who showed no response (P < 0.05). These findings suggest that a high serum sIL-2R level reflects the leukocyte growth in CMPD and is useful both for differentiating CML from other CMPD and for predicting the response to IFN therapy in CML. PMID:9071816

  3. Protein Phosphatase 2A Regulates Interleukin-2 Receptor Complex Formation and JAK3/STAT5 Activation*

    PubMed Central

    Ross, Jeremy A.; Cheng, Hanyin; Nagy, Zsuzsanna S.; Frost, Jeffrey A.; Kirken, Robert A.

    2010-01-01

    Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rβ, but not IL-2Rγ. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rβ. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rβ, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rβ by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rγ in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease. PMID:19923221

  4. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2.

    PubMed

    Clayton, Aled; Mitchell, J Paul; Court, Jacquelyn; Mason, Malcolm D; Tabi, Zsuzsanna

    2007-08-01

    Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4(+) T cells, CD8(+) T cells, and NK cells revealed that CD8(+) T-cell proliferation was not inhibited in the absence of CD4(+) T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2-mediated CD25 up-regulation, affecting all but the CD3(+)CD8(-) T-cell subset. IL-2-induced Foxp3 expression by CD4(+)CD25(+) cells was not inhibited by tumor exosomes, and the suppressive function of CD4(+)CD25(+) T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell-independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor beta(1) (TGFbeta(1)), which contributed to the antiproliferative effects, shown by using neutralizing TGFbeta(1)-specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated "double hit" to cellular immunity strongly implicates the role of exosomes in tumor immune evasion. PMID:17671216

  5. Soluble interleukin-2 receptors (sIL-2R) in Hodgkin's disease: outcome and clinical implications.

    PubMed Central

    Viviani, S.; Camerini, E.; Bonfante, V.; Santoro, A.; Balzarotti, M.; Fornier, M.; Devizzi, L.; Verderio, P.; Valagussa, P.; Bonadonna, G.

    1998-01-01

    The aim of this study was to assess the prognostic role of soluble interleukin-2 receptors (sIL-2R) in Hodgkin's disease (HD) both in the achievement of complete remission (CR) and in predicting disease relapse. Between August 1988 and June 1993 sIL-2R serum levels were measured in 174 untreated patients; in 137 of them evaluation was repeated at the end of treatment and in 132 also during the follow-up. Baseline sIL-2R levels (mean+/-standard error) were significantly higher in patients than in 65 healthy control subjects (1842+/-129 U ml(-1) vs 420+/-10 U ml(-10, P< 0.0001). At the end of treatment 135 out of 137 evaluated patients achieved complete response (CR) and their mean sIL-2R serum levels were significantly lower than those at diagnosis (635+/-19 U ml(-1) vs 1795+/-122 U ml(-1), P=0.0001). After a median follow-up of 5 years, sIL-2R remained low in 114 patients in continuous CR, while they increased in 9 out of 12 patients (75%) who relapsed. However, a temporary increase was also observed in six patients (5%) still in CR. Treatment outcome in terms of freedom from progression was linearly related to sIL-2R levels. Our study confirms that patients with untreated HD have increased baseline levels of sIL-2R compared with healthy subjects and that their pretreatment values may be an indication of disease outcome similar to other conventional prognostic factors, such as number of involved sites, presence of B symptoms and extranodal extent. PMID:9528846

  6. Bronchoalveolar lavage analysis, gallium-67 lung scanning and soluble interleukin-2 receptor levels in asbestos exposure.

    PubMed

    Delclos, G L; Flitcraft, D G; Brousseau, K P; Windsor, N T; Nelson, D L; Wilson, R K; Lawrence, E C

    1989-04-01

    This study examined different markers of lung immunologic and inflammatory responses to previous asbestos exposure. We performed bronchoalveolar lavage (BAL) and gallium-67 (67Ga) lung scans and measured serum and BAL soluble interleukin-2 receptor (IL-2R) and angiotensin-converting enzyme (SACE) levels in 32 subjects with a history of significant asbestos exposure, 14 without (EXP) and 18 with (ASB) radiographic evidence of asbestosis. BAL analysis revealed increases in neutrophils in both ASB and EXP when compared to controls (P less than 0.01), which persisted after adjustment for smoking category. Although significant abnormalities of macrophage and total lymphocyte profiles were not found in the study population, lymphocyte subpopulation analysis revealed elevation of BAL T4/T8 ratios in the entire study group (ASB + EXP) when compared to controls (P less than 0.05), independent of smoking category. 67Ga lung scan activity was increased in 56% of ASB and in 36% of EXP: no correlations between positive scans and different radiological and functional parameters could be found. There was no significant elevation of mean SACE, serum, or BAL IL-2R levels in any of the study categories. These data suggest that asbestos exposure may be associated with parenchymal inflammation, even in the absence of clinical criteria for asbestosis. Abnormalities of gallium uptake and of BAL analysis reflect the clinically inapparent inflammation. The increased BAL T4/T8 ratios observed suggest that abnormal local pulmonary immunoregulation may play a role in the pathogenesis of asbestos-related lung diseases. PMID:2538325

  7. Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

    SciTech Connect

    Atluru, D.; Polam, S.; Atluru, S. ); Woloschak, G.E. )

    1990-01-01

    Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, the authors examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-simulated ({sup 3}H)thymidine uptake, IL-2production, and IL-2R expression in a dose-related manner. Further, they found H-7 inhibited T-cell proliferation, IL-2 production, and IL-2mRNA from PHA plus PMA-stimulated cultures. They also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. The results demonstrate that PKC activation is one major pathway through which T-cells become activated.

  8. Successful Treatment of TAFRO Syndrome, a Variant of Multicentric Castleman's Disease, with Cyclosporine A: Possible Pathogenetic Contribution of Interleukin-2.

    PubMed

    Konishi, Yoshinobu; Takahashi, Satoshi; Nishi, Katsuyuki; Sakamaki, Taro; Mitani, Sachiko; Kaneko, Hitomi; Mizutani, Chisato; Ukyo, Naoya; Hirata, Hirokazu; Tsudo, Mitsuru

    2015-01-01

    Multicentric Castleman's disease is a systemic inflammatory disorder characterized by lymphadenopathy and excessive interleukin-6 production. A unique clinicopathologic variant of multicentric Castleman's disease, TAFRO (i.e., thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly) syndrome, was recently proposed in Japan. Despite the successful use of anti-interleukin-6 therapy in some patients with TAFRO syndrome, not all patients achieve remission. The pathophysiological etiology of and suitable therapeutic strategies for this variant have not been established. Here, we present our experience of a unique case of TAFRO syndrome in a 78-year-old woman whose symptoms responded differently to several therapies. Tocilizumab, an anti-interleukin-6 receptor antibody, successfully induced remission of fever and lymphadenopathy. However, severe thrombocytopenia persisted and she developed anasarca, ascites, and pleural effusion shortly thereafter. Rituximab, an anti-CD20 antibody, and glucocorticoid therapy provided no symptom relief. In contrast, cyclosporine A, an immunosuppressive agent that blocks T cell function by inhibiting interleukin-2, yielded immediate improvements in systemic fluid retention and a gradual increase in platelet count, with complete resolution of disease symptoms. Excessive serum interleukin-2, when used as an anti-cancer agent, has been reported to cause side effects such as fluid retention, thrombocytopenia, and renal failure. Our case was unique because the anti-interleukin-2 therapy successfully improved symptoms that were not relieved with anti-interleukin-6 therapy. The present report therefore provides insight into the possible role of interleukin-2, in addition to interleukin-6, in TAFRO syndrome. This report will certainly help to clarify the pathogenesis of and optimal treatment strategies for TAFRO syndrome. PMID:26250536

  9. Pulse infusion interleukin-2 with famotidine and cyclophosphamide has activity in previously treated metastatic melanoma.

    PubMed

    Quan, Walter; Knupp, Charles; Quan, Francine; Walker, Paul

    2010-04-01

    There is no established systemic therapy for patients with stage IV melanoma refractory to prior systemic treatment. Interleukin-2 (IL-2) is capable of inducing T-lymphocyte cytotoxicity against melanoma in vitro and in vivo. Famotidine may enhance the activity of T-cells further by allowing for increased IL-2 internalization by the IL-2 receptor on lymphocytes. Cyclophosphamide may decrease the immunosuppressive effects of regulatory T-cells. Daily short intravenous (i.v.) infusions (pulses) of IL-2 were used to treat 14 patients with metastatic melanoma, all of whom had experienced disease progression despite prior systemic therapy. The patients received 21.6 million IU/m(2) of pulse IL-2 i.v. for 15-30 minutes, preceded by 20 mg of famotidine i.v. (13) patients received 350 mg/m(2) of cyclophosphamide i.v. on day 1 (1 patient did not). Eight (8) patients were treated in an oncology inpatient unit while, most recently, 6 patients have received therapy on an outpatient basis. The cycles were repeated every 3 weeks until disease progression occurred. The patients included 10 males with a median age of 56 (range 31-87) with an Eastern Cooperative Oncology Group performance status of -1 (range 0 - -1). Common metastatice sites included lymph nodes (13), lungs (8), liver (4), and subcutaneous (4). Prior systemic therapy included IL-2 (11), interferon (7), and chemotherapy (7). The median number of cycles the patients underwent was 3 with a range of 1-7. The most common toxic reactions were fever, rigors, nausea/emesis, hypomagnesemia, and hypophosphatemia. One complete response and four partial responses were observed (response rate, 36%; 95% confidence interval: 14%-64%). Responses occurred in the lungs, liver, lymph nodes, and subcutaneous sites. The median response duration was 3.4 months, with a median survival of 8.3 months for the entire group. Six (6) patients remain alive with a median survival of 10.3 months. Pulse IL-2 with famotidine and cyclophosphamide

  10. Impaired left ventricular filling rate induced by treatment with recombinant interleukin 2 for advanced cancer.

    PubMed Central

    Fragasso, G.; Tresoldi, M.; Benti, R.; Vidal, M.; Marcatti, M.; Borri, A.; Besana, C.; Gerundini, P. P.; Rugarli, C.; Chierchia, S.

    1994-01-01

    BACKGROUND--Immunotherapy with recombinant interleukin 2 (rIL 2) has been extensively used to treat cancer but its use has been hampered by serious side effects including severe hypotension, arrhythmias, and myocardial infarction. OBJECTIVE--To assess the effects of rIL 2 on human left ventricular function. METHODS--Left ventricular (LV) function was monitored in 22 patients (9 women, 13 men) (mean (SD) age 53 (10) years) undergoing a 120 h continuous intravenous infusion of rIL 2 (18 x 10(6) IU/m2/day) for melanoma (4), renal cell (16), ovarian (1), and colon cancer (1). Radionuclide ventriculography was performed before and 1 h after the end of treatment. Ejection fraction (EF), peak emptying rate (PER), peak filling rate (PFR), and regional left ventricular wall motion were analysed. Heart rate (HR), central venous pressure (CVP), systolic (SBP) and diastolic blood pressures (DBP), the electrocardiogram, and myocardial enzyme concentrations were monitored throughout the study. RESULTS--All variables (mean (SD)) were normal before rIL 2 was given. After rIL 2 administration HR increased significantly from 84 (11) to 125 (18) beats/min (p < 0.0001), SBP fell from 128 (11) to 100 (9) mmHg (p < 0.001) and DBP from 76 (9) to 65 (7) mmHg (p < 0.0001). CVP decreased from 3.70 (3.2) to 1.30 (0.45) cm H2O (p < 0.001). EF (65 (7) to 64 (8%) and PER (3.56 (0.60) to 3.86 (0.83) EDV/s) did not change significantly. PFR decreased significantly at the end of the rIL 2 infusion from 2.68 (0.46) to 2.37 (0.43) EDV/s (p < 0.01). Left ventricular segmental hypokinesia developed in 6 patients. Myocardial enzyme concentrations remained normal throughout the study. CONCLUSIONS--The results of this study confirmed that rIL 2 produces important haemodynamic changes, predominantly related to decreased systemic resistance. However, the observed reduction in PFR in most patients suggested that rIL 2 might exert its action at the level of the heart muscle itself. The localised systolic

  11. Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line.

    PubMed Central

    Karnitz, L; Sutor, S L; Torigoe, T; Reed, J C; Bell, M P; McKean, D J; Leibson, P J; Abraham, R T

    1992-01-01

    The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, p56lck, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable lck-derived mRNA transcripts, protein, or PTK activity. The p56lck-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56lck activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56lck-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56lck-deficient cell line displayed a profound reduction in T-cell antigen receptor-dependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56lck-deficient cells were completely reversed by transfection of these cells with a wild-type lck expression vector. These results indicate that p56lck expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses. Images PMID:1406641

  12. Plasma nitrate plus nitrite changes during continuous intravenous infusion interleukin 2.

    PubMed Central

    Citterio, G.; Pellegatta, F.; Lucca, G. D.; Fragasso, G.; Scaglietti, U.; Pini, D.; Fortis, C.; Tresoldi, M.; Rugarli, C.

    1996-01-01

    Nitric oxide (NO), a biologically active mediator generated in many cell types by the enzyme NO synthase, may play an important role in cardiovascular toxicity that is frequently observed in cancer patients during intravenous (i.v.) interleukin 2 (IL-2) therapy. The induction of NO synthase and the production of NO seem to be involved in the pathogenesis of the vascular leakage syndrome, as well as in the regulation of myocardial contractility. In the present study, we evaluated the pattern of plasmatic NO changes during multiple cycles of continuous i.v. infusion (CIVI) of IL-2 in ten advanced cancer patients (five males, five females, median age 59 years, range 33-67 years; eight affected by renal cell cancer and two affected by malignant melanoma). The patients received IL-2 at 18 MIU m-2 day-1 (14 cycles) or 9 MIU m-2 day-1 (seven cycles) for 96 h, repeated every 3 weeks. Interferon alpha (IFN alpha) was also administered subcutaneously (s.c) during the 3 week interval between IL-2 cycles. For each cycle, plasma samples were collected before treatment (t0), 24 h (t1), 48 h (t2), 72 h (t3) and 96 h (t4) after the start of IL-2 infusion, and 24 h after the end of the cycle. NO concentration was determined spectrophotometrically by measuring the accumulation of both nitrite and nitrate (after reduction to nitrite). The following observations may be drawn from data analysis: (1) plasma nitrate + nitrite significantly raised during treatment (P = 0.0226 for t0 vs t3), but statistical significance was retained only when cycles administered with IL-2 18 MIU m-2 day-1 are considered (P = 0.0329 for t0 vs t3; P = 0.0354 for t0 vs t2 vs t4) (dose-dependent pattern); (2) during subsequent cycles a significant trend toward a progressive increase of plasma nitrate + nitrite levels, with increasing cumulative dose of IL-2, was observed (linear regression coefficient r = 0.62, P = 0.0141 for t0; r = 0.80, P = 0.0003 for t1; r = 0.62, P = 0.013 for t2; r = 0.69, P = 0.045 for

  13. T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

    PubMed Central

    Carel, S; Bron, C; Corradin, G

    1983-01-01

    T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2. Images PMID:6192442

  14. Six weeks of aerobic dance exercise improves blood oxidative stress status and increases interleukin-2 in previously sedentary women.

    PubMed

    Leelarungrayub, Donrawee; Saidee, Kunteera; Pothongsunun, Prapas; Pratanaphon, Sainetee; YanKai, Araya; Bloomer, Richard J

    2011-07-01

    This study evaluated the change in blood oxidative stress, blood interleukin-2, and physical performance following 6 weeks of moderate intensity and duration aerobic dance exercise in 24 sedentary women. Blood samples were collected at rest twice before (baseline) and after the 6-week intervention for analysis of protein hydroperoxide (PrOOH), malondialdehyde (MDA), total anti-oxidant capacity (TAC), and interleukin-2 (IL-2) levels. Maximal treadmill run time (Time(max)) and maximal oxygen consumption (VO(2max)) were also measured. All variables were statistically analyzed with a repeated measurement ANOVA and Tukey post hoc. No differences were noted in any variable during the baseline period (p > 0.05). After aerobic dance exercise, VO(2max), Time(max), TAC and IL-2 were significantly increased, whereas MDA levels were decreased significantly (p < 0.05). PrOOH did not change either between baseline measures or after exercise. It can be concluded that aerobic dance exercise at a moderate intensity and duration can improve physical fitness, decrease MDA, and increase TAC and IL-2 in previously sedentary women. PMID:21665113

  15. 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.

  16. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  17. TSC-22 Promotes Interleukin-2-Deprivation Induced Apoptosis in T-Lymphocytes.

    PubMed

    Pépin, Aurélie; Espinasse, Marie-Alix; Latré de Laté, Perle; Szely, Natacha; Pallardy, Marc; Biola-Vidamment, Armelle

    2016-08-01

    Originally described as a TGF-β-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-β. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc. PMID:26752201

  18. Nursing care of patients receiving high-dose, continuous-infusion interleukin-2 with pulse dose and famotidine.

    PubMed

    Tyre, Charley Cowan; Quan, Walter

    2007-08-01

    High-dose, continuous-infusion interleukin-2 (IL-2) followed by pulse dose and concurrent administration of famotidine has demonstrated response rates of 64% and 33% in patients with metastatic melanoma and metastatic renal cell carcinoma, respectively. Currently, no information is available concerning the nursing care of patients receiving that IL-2 regimen. Given the high response rates of patients on the treatment, attention by the nursing profession is warranted. Effective nursing care of patients receiving IL-2 is essential to the regimen's success. Recognition and prompt treatment of common side effects lead to better patient outcomes. This article provides nurses with an overview of the treatment regimen, expected side effects, psycho-social considerations, and discharge instructions for patients receiving continuous-infusion plus pulse IL-2 and famotidine. PMID:17723964

  19. The Toxicity and Benefit of Various Dosing Strategies for Interleukin-2 in Metastatic Melanoma and Renal Cell Carcinoma

    PubMed Central

    Pachella, Laura A.; Madsen, Lydia T.; Dains, Joyce E.

    2015-01-01

    Interleukin-2 (IL-2) therapy has been used with success in curing meta­static renal cell carcinoma and melanoma in a small minority of patients. However, the benefits can be accompanied by severe toxicity. This review of the literature discusses varying doses of IL-2 and their associated re­sponse rates and the toxicities associated with treatment. The review also explores the maximally beneficial dose with the most tolerable side effects. Although the higher-dose regimens with a more frequent dosing schedule produce higher-grade toxicity, they were found to deliver the most durable and complete responses. It is recommended to use a higher-dose regimen (720,000 IU/kg every 8 hours for a maximum of 15 doses) and provide sup­portive care for toxicity, so patients can have maximal benefit from therapy. PMID:26557408

  20. The Toxicity and Benefit of Various Dosing Strategies for Interleukin-2 in Metastatic Melanoma and Renal Cell Carcinoma.

    PubMed

    Pachella, Laura A; Madsen, Lydia T; Dains, Joyce E

    2015-01-01

    Interleukin-2 (IL-2) therapy has been used with success in curing meta-static renal cell carcinoma and melanoma in a small minority of patients. However, the benefits can be accompanied by severe toxicity. This review of the literature discusses varying doses of IL-2 and their associated re-sponse rates and the toxicities associated with treatment. The review also explores the maximally beneficial dose with the most tolerable side effects. Although the higher-dose regimens with a more frequent dosing schedule produce higher-grade toxicity, they were found to deliver the most durable and complete responses. It is recommended to use a higher-dose regimen (720,000 IU/kg every 8 hours for a maximum of 15 doses) and provide sup-portive care for toxicity, so patients can have maximal benefit from therapy. PMID:26557408

  1. Immunotherapy of murine sarcomas with interleukin 2. II. Activation of killer cells by human recombinant IL-2.

    PubMed

    Indrová, M; Bubeník, J; Toulcová, A

    1986-01-01

    Highly purified human recombinant interleukin 2 induced cytotoxicity in mouse spleen cells against mouse sarcoma cells when added during the 51Cr microcytotoxicity assay. It elicited similar levels of killer cell activation as did human lymphoid (Jurkat leukaemia-derived) or mouse lymphoid (EL-4 leukaemia-derived) IL-2 preparations. The susceptibility of six MC-induced mouse sarcomas to the cytolytic effect of lymphokine-activated killer cells was compared. Five (MC11, MC13, MC14, MC15, MC16) of six mouse sarcoma cell lines examined were sensitive in vitro to the LAK cell effect, whereas one cell line (MC12) was resistant. Since the sensitive and resistant target cell lines had been induced with the same carcinogen and in mice of the same genotype, they represent a very useful model for investigation of target cell structures responsible for the sensitivity to the LAK cell effect. PMID:3492397

  2. Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+ T cell expansion in an immunocompetent mouse model.

    PubMed

    Foureau, David M; McKillop, Iain H; Jones, Chase P; Amin, Asim; White, Richard L; Salo, Jonathan C

    2011-09-01

    Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant responses are observed in only 10-15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB/MS, MCA-205) and nonresponsive (JB/RH, B16-F10) subcutaneous tumor mouse models, we evaluated CD8 Foxp3+ T cell distribution and changes in response to rhIL-2 (50,000 U, i.p. or s.q., twice daily for 5 days). In tumor-free mice and subcutaneous tumor-bearing mouse models, CD8 Foxp3+ T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes, CD8 Foxp3+ T cells expressed both activated T cell (CD28(+), CD44(+)) and Treg (CTLA4(+), PD1(lo/var), NKG2A(+/var)) markers. Following treatment with rhIL-2, a dramatic increase in CD8 Foxp3+ T cell prevalence was observed in the circulation and tumor-draining lymph nodes (TD.LNs) of animals bearing IL-2 nonresponsive tumors, while no significant changes were observed in the circulation and TD.LNs of animals bearing IL-2 responsive tumors. These findings suggest expansion of CD8 Foxp3+ T cell population in response to rhIL-2 treatment may serve as an early marker for tumor responsiveness to immunotherapy in an immune competent model. Additionally, these data may provide insight to predict response in patients with melanoma undergoing rhIL-2 treatment. PMID:21638127

  3. Synergy between interleukin-2 and a second factor in the long-term growth of human T cells.

    PubMed Central

    Mills, B J; Shively, J E; Mitsky, P S; Hawke, D H; Forman, S J; Wright, C L; Todd, C W

    1986-01-01

    It has recently been shown that factors in addition to interleukin-2 (IL-2) are required for the proliferation or differentiation of at least some murine T-cell lines. We have previously shown that conditioned medium from human mononuclear cells stimulated with phorbol ester and staphylococcal enterotoxin A is superior to commercial sources of IL-2 for the long-term growth of human T cells. We have identified in these supernatants a non-IL-2 factor (synergistic factor, SF) which synergizes with JURKAT IL-2 in the long-term growth of human T cells. [3H]TdR incorporation by IL-2-dependent human T cells after growth in IL-2 or SF alone for 14 days was slight, but significant. By contrast, growth in a combination of SF and IL-2 for 14 days stimulated [3H]TdR incorporation 10-20-fold higher, generally equal to the high incorporation measured when cells were grown in the presence of the conditioned medium from which SF was obtained. In a standard 2-day IL-2 assay, there was no correlation between activity and long-term growth-promoting ability. These results suggest that the 14-day assay better discerns the growth-promoting activity of various factors or combinations of factors. The mechanism of this interaction between SF and IL-2 remains to be elucidated. It is clear, however, that T-cell growth factor activity, when assessed by the long-term growth of human T cells, is not due to interleukin-2 alone. PMID:3489670

  4. 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...

  5. Unfractionated human thymocytes have a lower proliferative capacity than CD3/sup -/4/sup -/8/sup -/ ones but have a similar capacity for expression of interleukin 2 receptors and production of interleukin 2

    SciTech Connect

    Vives, J.; Sole, J.; Suarez, B.

    1987-12-01

    CD3/sup -/4/sup -/8/sup -/ and unfractionated thymocytes were compared for their capacity to proliferate, to express interleukin 2 (IL-2) receptor, and to secrete IL-2. Phorbol ester and Ca/sup 2 +/ ionophore were used as mitogens. CD3/sup -/4/sup -/8/sup -/ thymocytes responded vigorously when stimulated with phorbol ester in the presence of IL-2 or in combination with Ca/sup 2 +/ ionophore. In contrast, unfractionated thymocytes responded weakly when stimulated with either of these mitogens. Surprisingly, however, the stimulation of these populations with either phorbol ester plus IL-2 or phorbol ester plus ionophore induced a high and similar level of IL-2 receptor expression in both thymocyte populations. A similar level of IL-2 secretion in both populations was also obtained when they were stimulated with a combination of phorbol ester plus ionophere. These results suggest that during the maturation process, the majority of thymocytes lose their capacity to be activated by some mitogens, although they maintain their capacity to secrete IL-2 and to express the IL-2 receptor.

  6. Immunomodulatory therapy with thymopentin and indomethacin. Successful restoration of interleukin-2 synthesis in patients undergoing major surgery.

    PubMed Central

    Faist, E; Markewitz, A; Fuchs, D; Lang, S; Zarius, S; Schildberg, F W; Wachter, H; Reichart, B

    1991-01-01

    Prostaglandin E2 (PGE2)-mediated monocyte (M phi) suppressor activity and inadequate T-helper cell function represent the mechanistic keystones of trauma-induced impairment of cell-mediated immunity (CMI). In a prospective randomized trial, the immunorestorative potential of a combined therapy with the thymomimetic substance Thymopentin (TP-5; Timunox, Cilag GMBH, Sulzbach, FRG) and the cyclooxygenase inhibitor indomethacin (Indo) in 60 patients (mean age, 63 +/- 2 years) undergoing open heart surgery was studied. Perioperative immunologic screening was carried out on days -2, 3, 1, 5, and 7 and included the in vivo delayed type hypersensitivity (DTH) skin response, phenotyping for peripheral blood mononuclear cell (PBMC)-specific and nonspecific induction of lymphoproliferative responses, in vitro interleukin-2 (IL-2) synthesis, as well as the serum concentration of D-erythro-Neopterin (NPT) and of gamma interferon (gamma-IFN). The study protocol comprised three groups (n = 20): PA (Indo 150 mg administered intravenously on days 0 to 5), PB (TP-5 administered subcutaneously on days 0, 2, 4, and Indo), and PC (control). In contrast to PC, significant immunorestoration could be demonstrated in PB, as DTH scores on day 7, as well as proliferative responses in cell cultures were not depressed after operation (p less than 0.05). Cell-surface receptor expression for the CD3+, CD4+, and IL-2 receptor-positive (IL-2R+) lymphocyte subpopulations following surgery was reduced to 75% of baseline values in PC, while in PB, receptor protection for CD4+ and IL-2R+ subpopulations (more than 15% above baseline) was observed. Interleukin-2 synthesis (average baseline value, 0.7 + 0.08 U/mL) in cell cultures of PC was massively suppressed, with lymphokine concentrations in the supernatants never more than 0.27 +/- 0.05 U/mL. In PA cultures, IL-2 synthesis was impaired as well but not as precipitously as in PC. In contrast, in PB cultures, the average IL-2 production on consecutive

  7. Subcutaneous administration of interleukin 2 and interferon-alpha-2b in advanced renal cell carcinoma: a confirmatory study.

    PubMed Central

    Facendola, G.; Locatelli, M. C.; Pizzocaro, G.; Piva, L.; Pegoraro, C.; Pallavicini, E. B.; Signaroldi, A.; Meregalli, M.; Lombardi, F.; Beretta, G. D.

    1995-01-01

    Recent clinical studies have suggested that the combination of subcutaneous recombinant human interleukin 2 (rIL-2) and interferon alpha (rIFN-alpha) is especially promising in advanced renal cell carcinoma. We assessed the safety, activity and toxicity of home therapy with these two agents in 50 patients. Each treatment cycle consisted of a 2 day pulse phase, with 9 x 10(6) IU m-2 of rIL-2 being given subcutaneously every 12 h, followed by a 6 week maintenance phase during which rIL-2 1.8 x 10(6) IU m-2 was administered subcutaneously every 12 h on days 1-5 and rIFN-alpha 2b 5 x 10(6) IU m-2 once a day on days 1, 3 and 5. Objective responses (CR+PR) occurred in 9/50 (18%) patients, six of whom (12%) achieved a complete response. Disease stabilisation was observed in 17 cases (34%) and 18 patients progressed during therapy. In the other six cases, treatment was interrupted early for toxicity or patient refusal. One patient died of myocardial infarction during the second cycle. The overall median survival was 12 months. Home therapy with subcutaneous rIL-2 + rIFN-alpha 2b proved to be active, feasible and moderately toxic, but serious adverse events can sometimes occur. PMID:8519672

  8. [Effect of acupuncture on interleukin-2 level and NK cell immunoactivity of peripheral blood of malignant tumor patients].

    PubMed

    Wu, B; Zhou, R X; Zhou, M S

    1994-09-01

    This paper deals with the observation of acupuncture therapy affecting interleukin-2(IL-2 level and natural killer (NK) cell immunoactivity in the peripheral blood of patients with malignant tumors. In this clinical-laboratory test research, randomized double blind method was used. The patients were divided into an acupuncture treated group (n = 25) and a control group (n = 20). The former group was treated using points, ST36,LI11,RN6 and locations of symptomatic points bilaterally. They received one treatment of 30 minutes daily for 10 days. The results showed that the IL-2 level and NK cell activity were lower than normal in patients with malignant tumor, but there was an increase in the acupuncture group after 10 days of treatment. Significance was found to be remarkable (P < 0.01). The difference between the two groups was also significant (P < 0.01). This increase might be related to the mechanism of acupuncture that adjusting the body's immune function. Thus, acupuncture therapy could enhance the cellular immune function of patients with malignant tumors and providing a beneficial effect in anti-cancer treatment. PMID:7866002

  9. 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.

  10. Use of interleukin-2 for management of natalizumab-associated progressive multifocal leukoencephalopathy: case report and review of literature

    PubMed Central

    Dubey, Divyanshu; Zhang, Yinan; Graves, Donna; DeSena, Allen D.; Frohman, Elliot; Greenberg, Benjamin

    2015-01-01

    A 51-year-old woman with relapsing–remitting multiple sclerosis (RRMS) and 3-year history of natalizumab use developed expressive aphasia. A brain magnetic resonance image (MRI) showed left frontotemporal and right parietal lesion with mild contrast enhancement and cerebrospinal fluid (CSF) was positive for John Cunningham virus (JCV) by polymerase chain reaction (PCR). The patient received five cycles of plasmapheresis followed by intravenous immunoglobulin. Despite this intervention, her speech deteriorated and she developed right hemiparesis. Upon referral to our institution, CSF quantitative JCV PCR was notable for 834 copies/ml. The patient was given an initial dose of 50,000 units of interleukin-2 (IL-2) subcutaneously (SQ) followed by 1 million units IL-2 SQ daily. Due to concern for immune reconstitution inflammatory syndrome (IRIS), the patient also received intravenous methylprednisone weekly. The regimen was tolerated well by the patient with no severe adverse effects. Clinically, the patient showed some improvement, and became more responsive and regained right lower extremity antigravity strength. After 12 weeks of IL-2 therapy, JCV quantitative PCR was notable for 31 copies/ml and the patient was more responsive. Due to persistence of JCV, IL-2 therapy was changed to mefloquine. At follow up after 6 months, the patient showed no clinical deterioration. PMID:27134676

  11. Use of interleukin-2 for management of natalizumab-associated progressive multifocal leukoencephalopathy: case report and review of literature.

    PubMed

    Dubey, Divyanshu; Zhang, Yinan; Graves, Donna; DeSena, Allen D; Frohman, Elliot; Greenberg, Benjamin

    2016-05-01

    A 51-year-old woman with relapsing-remitting multiple sclerosis (RRMS) and 3-year history of natalizumab use developed expressive aphasia. A brain magnetic resonance image (MRI) showed left frontotemporal and right parietal lesion with mild contrast enhancement and cerebrospinal fluid (CSF) was positive for John Cunningham virus (JCV) by polymerase chain reaction (PCR). The patient received five cycles of plasmapheresis followed by intravenous immunoglobulin. Despite this intervention, her speech deteriorated and she developed right hemiparesis. Upon referral to our institution, CSF quantitative JCV PCR was notable for 834 copies/ml. The patient was given an initial dose of 50,000 units of interleukin-2 (IL-2) subcutaneously (SQ) followed by 1 million units IL-2 SQ daily. Due to concern for immune reconstitution inflammatory syndrome (IRIS), the patient also received intravenous methylprednisone weekly. The regimen was tolerated well by the patient with no severe adverse effects. Clinically, the patient showed some improvement, and became more responsive and regained right lower extremity antigravity strength. After 12 weeks of IL-2 therapy, JCV quantitative PCR was notable for 31 copies/ml and the patient was more responsive. Due to persistence of JCV, IL-2 therapy was changed to mefloquine. At follow up after 6 months, the patient showed no clinical deterioration. PMID:27134676

  12. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    PubMed Central

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  13. Interleukin-2 protects neonatal mice from lethal herpes simplex virus infection: a macrophage-mediated, gamma interferon-induced mechanism.

    PubMed

    Kohl, S; Loo, L S; Drath, D B; Cox, P

    1989-02-01

    Administration of human recombinant interleukin-2 (IL-2) protected neonatal mice from a lethal herpes simplex virus (HSV) infection. Protection was not associated with viral antibody production, enhanced natural killer cell cytotoxicity, or intrinsic resistance of macrophages to viral infection. Protection was associated with increased macrophage-mediated antiviral antibody-dependent cellular cytotoxicity (ADCC). Spleen cells from IL-2-treated neonatal mice and from neonatal mice that were treated in vitro with IL-2 transferred protection to neonatal mice. These cells, by adherence, silica, and asialo GM 1 antibody treatment, were shown to be macrophages. IL-2 treatment in vitro enhanced the neonatal macrophages' ADCC function and superoxide release. Similar protection was induced by gamma interferon (IFN-gamma)-treated spleen cells. Antibody to IFN-gamma ablated both IFN-gamma- and IL-2-induced protection by adherent spleen cells. Thus, IL-2-mediated protection against murine neonatal HSV infection was affected by stimulated macrophage activity, via helper T cell-produced IFN-gamma. PMID:2492588

  14. A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

    PubMed

    Shanafelt, A B; Lin, Y; Shanafelt, M C; Forte, C P; Dubois-Stringfellow, N; Carter, C; Gibbons, J A; Cheng, S L; Delaria, K A; Fleischer, R; Greve, J M; Gundel, R; Harris, K; Kelly, R; Koh, B; Li, Y; Lantz, L; Mak, P; Neyer, L; Plym, M J; Roczniak, S; Serban, D; Thrift, J; Tsuchiyama, L; Wetzel, M; Wong, M; Zolotorev, A

    2000-11-01

    Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS. PMID:11062441

  15. Reduced secondary cytokine induction by BAY 50-4798, a high-affinity receptor-specific interleukin-2 analog.

    PubMed

    Steppan, Sonja; Eckart, Michael R; Bajsarowicz, Krystyna; Sternberg, Lawrence R; Greve, Jeffrey M; Cassell, Delanie J

    2006-03-01

    Recombinant interleukin-2 (IL-2) (aldesleukin, Proleukin, Chiron, Emeryville, CA) is approved for treatment of cancer patients and under investigation in HIV-infected individuals. However, treatment with aldesleukin is associated with toxicity, which may be due to its elicitation of inflammatory mediators from cells that express the intermediate-affinity IL-2 receptor. BAY 50-4798, a novel IL-2 analog, is a selective agonist for the high-affinity receptor. It induces the proliferation of activated T cells with a potency similar to that of aldesleukin but has reduced activity on cells expressing the intermediate-affinity receptor. In the current study, we compared cytokine responses elicited in peripheral blood mononuclear cell (PBMC) cultures stimulated with BAY 50-4798 or aldesleukin. BAY 50-4798 induced approximately 5-fold lower mean levels of endogenous IL-2 than aldesleukin, and at least 50% lower levels of proinflammatory cytokines, such as tumor necrosis fctor-alpha (TNF-alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma). Furthermore, statistically significant reductions in the levels of IL-5, IL-8, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed in response to BAY 50-4798. These findings increase our understanding of the biologic action of BAY 50-4798 and suggest a mechanism by which it may exhibit better safety than aldesleukin in humans. PMID:16542139

  16. Regression of metastatic clear cell kidney cancer with interleukin-2 treatment following nivolumab (anti-PD-1) treatment.

    PubMed

    Brayer, Jason; Fishman, Mayer

    2014-04-01

    Aldesleukin [interleukin-2 (IL-2)] induces durable complete responses in some kidney cancer and melanoma patients. Nivolumab is an investigational antibody drug targeting programmed death-1 (PD-1) as a treatment, demonstrating activity in multiple cancer types. An expanding complement of immunotherapeutics raises important issues regarding the best way to use them. There are issues beyond identifying an agent that provides the superior front-line response: when does one therapy potentiate another immune therapy? When is the capacity of immune response exhausted and an approach without immune mechanism the better therapy? In this case report, we present a patient with metastatic renal cell carcinoma with no tumor regression evident on a PD-1 blockade (given on an investigational trial), who then achieved near-complete response to bolus high-dose IL-2 therapy, maintaining a persistent response off therapy. This case emphasizes on the need to develop improved predictors of response to immune therapies, especially as they can be applied to optimize sequential immunotherapeutic modalities versus predict when to turn to alternative targeted agents in renal cell carcinoma, and is an example of efficacious IL-2 application as a second-line treatment. PMID:24598453

  17. Combined levamisole with recombinant interleukin-2 (IL-2) in patients with advanced renal cell carcinoma: a phase II study.

    PubMed

    Creagan, E T; Hestorff, R D; Suman, V J; Mailliard, J A; Nair, S; Krook, J E; Kugler, J W; Marschke, R F; Michalak, J C; Tschetter, L K

    1998-04-01

    Adoptive immunotherapy (AI) with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells is an antineoplastic modality in which immune-activated cells are administered to a host having cancer in an attempt to mediate tumor regression. Levamisole (LEV), an immune stimulant, has been suggested as having therapeutic effectiveness in a variety of cancers. After a phase I trial of recombinant IL-2 plus LEV, a phase II trial of this combination was conducted in patients who had advanced renal cell carcinoma. The regimen was IL-2 at 3 x 10(6) U/m2 daily x 5 plus LEV at 50 mg/m2 perorally three times a day x 5. Only one of the 22 eligible patients had a regression. It was a partial regression, 85 days in duration. The median time to treatment failure (refusal, progression, or off study because of toxicity) was 36 days. The only grade 4 toxicity reported was lethargy. This regimen is not recommended for further testing in patients who have advanced renal cell carcinoma. PMID:9537198

  18. 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 12.5°C 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 23.3°C 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

  19. Lymphokine-activated killer (LAK) cell phenomenon in cluster headache. "In vitro" activation by recombinant interleukin-2.

    PubMed

    Giacovazzo, M; Stirparo, G; DeStefano, L; Martelletti, P; Rinaldi-Garaci, C

    1989-03-01

    Previous studies showed that the Natural Killer (NK) activity of peripheral blood lymphocytes (PBL) from cluster headache (CH) patients is lower than that of controls. This decreased activity seems to be independent of the cluster period. beta-interferon has been shown to be more effective in increasing NK activity when incubated with PBL from CH patients, than with PBL from control donors. Lymphokine-Activated Killer (LAK) cells can be generated by incubation of human PBL in recombinant Interleukin-2 (rIL-2). This phenomenon was studied in 10 CH patients and 8 healthy volunteers. PBL were activated to LAK cells by "in vitro" incubation for 72 hours in Control Medium containing rIL-2 (1000 I.U./ml). A four hour Chromium 51 release was used to measure LAK Cell Killing of K562 target cells. The released radioactivity was measured in a gamma scintillation counter. The CH patients showed a marked increase of LAK generation compared to control subjects. This effect seems to be augmented during the cluster period. PMID:2785095

  20. 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

  1. 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 43°C 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

  2. Functional impairment of natural killer cells in active ulcerative colitis: reversion of the defective natural killer activity by interleukin 2.

    PubMed Central

    Manzano, L; Alvarez-Mon, M; Abreu, L; Antonio Vargas, J; de la Morena, E; Corugedo, F; Duràntez, A

    1992-01-01

    We have studied the functional characteristics and clinical importance of the natural killer (NK) cytotoxicity of peripheral blood mononuclear cells (PBMNC) from patients with ulcerative colitis. Normal NK activity was observed in PBMNC from patients with inactive disease, but a pronounced decrease was found in those with active disease. Clinical change from active to inactive disease was associated with enhancement of the depressed NK activity. The impairment of NK cytotoxicity found in patients with active disese could not be ascribed to a deficient number of NK cells as the amounts of HNK-1+, CD16+ (Leu 11), and CD11b (OKM1) cells in PBMNC were within normal ranges. This defective cytotoxic PBMNC activity was normalised by short term (18 hour) incubation with recombinant interleukin 2 (rIL-2). Moreover, long term (5 day) incubation of these effector cells with rIL-2 induced strong cytotoxic activity against NK resistant and NK sensitive target cells in patients with active and inactive disease. We also found that both precursors and effectors of cytotoxic activity promoted by short term and long term incubation with rIL-2 of PBMNC from the patients showed the phenotype of NK cells (CD16+, CD3-). Taken together, these results show that active ulcerative colitis is associated with a defective function of NK cells that is found to be normal in the inactive stage of the disease. The possible pathogenic and therapeutic implications of these findings are discussed. PMID:1541421

  3. Evidence of enhanced recombinant interleukin-2 sensitivity in thymic lymphocytes from patients with myasthenia gravis: possible role in autoimmune pathogenesis.

    PubMed

    Cohen-Kaminsky, S; Levasseur, P; Binet, J P; Berrih-Aknin, S

    1989-09-01

    We evaluated the activation state of thymic lymphocytes in patients with myasthenia gravis (MG) by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant interleukin-2 (rIL-2) in the absence of any known previous stimulation. We detected no phenotypic signs of activation in fresh MG thymic lymphocyte suspensions, while functional signs of activation were reflected in a significantly higher sensitivity to rIL-2 in MG patients than in controls. The responses to rIL-2 were time- and dose-dependent, were inhibited by a blocking anti-IL-2 receptor antibody, and were associated with an increase in CD25+ T cells in both patients and controls. The T cells with functional signs of previous activation may represent autoreactive cells involved in the autoimmune process and confirm thymus gland hyperactivity in MG. These cells could result from primary autosensitization against the thymic acetylcholine receptor (AChR)-like molecule or from altered migration of peripheral activated cells into an abnormal thymic environment. Our results also provide a clue for understanding the effect of thymectomy in myasthenia gravis. PMID:2808688

  4. Effect of various mouthwashes on the levels of interleukin-2 and interferon-gamma in chronic gingivitis.

    PubMed

    Sharma, Shivalal; Saimbi, C S; Koirala, Bandana; Shukla, Rakesh

    2008-01-01

    The aim of this double blind study was to evaluate the effect of various mouthwashes: Chlorhexidine, Essential oil, Azadirachta indica (Neem) extract, and Povidone iodine on gingival tissue interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in patients with chronic gingivitis. A total of 8O patients (42 boys, 38 girls; mean age 16.0 +/- 1.8 years) were included in this study. Patients were randomly assigned into four groups of 20 each: Group I--Azadirachta indica (Neem) extract, Group II--Essential oil, Group III--Povidone iodine, and Group IV--Chlorhexidine. They were instructed to use these mouthwashes for two weeks. Plaque and gingival indices scores, and IL-2 and IFN-gamma levels in the gingival tissues were measured at baseline and after two weeks of mouthwash use. Results showed the reduction of plaque and gingival indices, and IL-2 and IFN-gamma level with Chlorhexidine, Essential oil, and Povidone iodine, which were found to be statistically significant. Although Neem reduced the level of plaque and gingival indices, and IL-2 and IFN-gamma to a certain level, it was not statistically significant. Therefore, Chlorhexidine, Essential oil, and Povidone iodine mouthwashes can be used as an adjunct to oral prophylaxis in reducing pro-inflammatory cytokines, IL-2 and IFN-gamma in patients with chronic gingivitis. PMID:18389675

  5. 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.

  6. Serum concentrations of soluble interleukin 2 receptor in patients with rheumatoid arthritis: effect of second line drugs.

    PubMed

    Crilly, A; Madhok, R; Watson, J; Capell, H A

    1993-01-01

    Serum soluble interleukin 2 receptor (sIL-2R) concentrations reflect lymphocyte activation in vivo. An investigation was carried out to determine if sIL-2R concentrations correlate with existing disease activity parameters in patients with rheumatoid arthritis (RA) and whether these concentrations are modulated by treatment with second line drugs. Seventy nine patients with rheumatoid arthritis with active disease were prospectively treated with sodium aurothiomalate, auranofin, or sulphasalazine. Sequential concentrations of sIL-2R were measured by enzyme linked immunosorbent assay (ELISA). No correlations were observed between sIL-2R concentrations and clinical parameters and there were only moderate associations with concentrations of C reactive protein and the erythrocyte sedimentation rate. Concentrations of sIL-2R did not significantly change with treatment. It is concluded that sIL-2R probably measures an aspect of rheumatoid synovitis distinct from acute phase reactants and is not influenced by treatment with second line drugs. PMID:8093995

  7. Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

    PubMed

    Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

    2005-12-15

    An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin. PMID:16112745

  8. Qualitative Immune Modulation by Interleukin-2 (IL-2) Adjuvant Therapy in Immunological Non Responder HIV-Infected Patients

    PubMed Central

    Sabbatini, Francesca; Bandera, Alessandra; Ferrario, Giulio; Trabattoni, Daria; Marchetti, Giulia; Franzetti, Fabio; Clerici, Mario; Gori, Andrea

    2010-01-01

    Background Treatment of HIV-infected patients with interleukin-2 (IL-2) produces significant increases in CD4 T cell counts; however an associated qualitative improvement in cells function has yet to be conclusively demonstrated. By measuring mycobacterial killing activity, we evaluated IL-2-mediated functional immune enhancement ex vivo in immunological non-responders (INRs). Methods and Findings PBMC from 12 immunological non-responders (INRs) (CD4+<200/µl, HIV-RNA<50 cp/ml) on combination antiretroviral treatment (cART) were collected at baseline, and after 3 IL-2 cycles. Eight INRs receiving only cART were studied as controls. After 21 days of PBMC incubation with a virulent M. avium suspension, counts of residual colony forming units (CFUs) and concentrations of TNF-α, IL-10 and IFN-γ were determined. In IL-2 treated patients, a significant reduction in mean residual CFUs of PBMC cultures was observed (p<0.01). Moreover, following IL-2 treatment, significant increases in PBMC's IFNγ production (p = 0.02) and substantial reductions in IL-10 levels were observed. Conclusions IL-2 therapy restores the ability of the lympho-monocyte system in eliciting an effective response against mycobacterial infections. Our data indicate the possibility of a clinical role held by IL-2 in enhancing the immune function of subjects unable to achieve immune competence through cART alone. PMID:21124762

  9. A phase II trial of trastuzumab in combination with low-dose interleukin-2 (IL-2) in patients (PTS) with metastatic breast cancer (MBC) who have previously failed trastuzumab.

    PubMed

    Mani, Aruna; Roda, Julie; Young, Donn; Caligiuri, Michael A; Fleming, Gini F; Kaufman, Peter; Brufsky, Adam; Ottman, Susan; Carson, William E; Shapiro, Charles L

    2009-09-01

    Trastuzumab mediates the lysis of HER2-expressing breast cancer cell lines by interleukin-2 (IL-2) primed natural killer (NK) cells. We hypothesized that IL-2 would augment the anti-tumor effects of trastuzumab in MBC in patients who had progressed on or within 12 months of receiving a trastuzumab-containing regimen. Secondary objectives were to measure antibody-directed cellular cytotoxicity (ADCC) against HER2 over-expressing target cells, and to measure serum cytokines. Patients received trastuzumab (4 mg/kg intravenously (IV)) every 2 weeks in combination with daily low-dose IL-2 (1 million IU/m(2) subcutaneously (SC)) and pulsed intermediate-dose IL-2 (12 million IU/m(2) SC). Samples were analyzed for NK cell expansion and ADCC against a HER2-positive breast cancer cell line. In addition, interferon-gamma (IFN-gamma), mRNA expression in peripheral blood mononuclear cells (PBMC) and the following serum cytokines were measured: IFN-gamma, monokine-induced by IFN-gamma (MIG), and interferon-inducible protein ten (IP-10). The median number of treatment cycles was four (range 1-23) and the treatment was well tolerated. There were no objective responses. NK cells were not expanded and ADCC was not enhanced. Eight (62%) patients had a twofold or higher increase in mRNA transcript for IFN-gamma, two (15%) patients had elevated serum levels of IFN-gamma and 12 (92%) had increases angiogenic MIG and IP-10. In trastuzumab-refractory patients adding IL-2 did not produce responses and did not result in NK cell expansion. However, these patients had the ability to respond to IL-2 as evidenced by increases in IFN-gamma transcripts and chemokines. The lack of NK cell expansion may explain the absence of clinical benefit. PMID:19051009

  10. A phase II trial of trastuzumab in combination with low-dose interleukin-2 (IL-2) in patients (PTS) with metastatic breast cancer (MBC) who have previously failed trastuzumab

    PubMed Central

    Mani, Aruna; Roda, Julie; Young, Donn; Caligiuri, Michael A.; Fleming, Gini F.; Kaufman, Peter; Brufsky, Adam; Ottman, Susan; Carson, William E.

    2010-01-01

    Trastuzumab mediates the lysis of HER2-expressing breast cancer cell lines by interleukin-2 (IL-2) primed natural killer (NK) cells. We hypothesized that IL-2 would augment the anti-tumor effects of trastuzumab in MBC in patients who had progressed on or within 12 months of receiving a trastuzumab-containing regimen. Secondary objectives were to measure antibody-directed cellular cytotoxicity (ADCC) against HER2 over-expressing target cells, and to measure serum cytokines. Patients received trastuzumab (4 mg/kg intravenously (IV)) every 2 weeks in combination with daily low-dose IL-2 (1 million IU/m2 subcutaneously (SC)) and pulsed intermediate-dose IL-2 (12 million IU/m2 SC). Samples were analyzed for NK cell expansion and ADCC against a HER2-positive breast cancer cell line. In addition, interferon-gamma (IFN-γ), mRNA expression in peripheral blood mononuclear cells (PBMC) and the following serum cytokines were measured: IFN-γ, monokine-induced by IFN-γ (MIG), and interferon-inducible protein ten (IP-10). The median number of treatment cycles was four (range 1–23) and the treatment was well tolerated. There were no objective responses. NK cells were not expanded and ADCC was not enhanced. Eight (62%) patients had a twofold or higher increase in mRNA transcript for IFN-γ, two (15%) patients had elevated serum levels of IFN-γ and 12 (92%) had increases angiogenic MIG and IP-10. In trastuzumab-refractory patients adding IL-2 did not produce responses and did not result in NK cell expansion. However, these patients had the ability to respond to IL-2 as evidenced by increases in IFN-γ transcripts and chemokines. The lack of NK cell expansion may explain the absence of clinical benefit. PMID:19051009

  11. Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex.

    PubMed Central

    Malek, T R; Robb, R J; Shevach, E M

    1983-01-01

    Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on greater than 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor. Images PMID:6412230

  12. Exogenous Interleukin-2 Administration Corrects the Cell Cycle Perturbation of Lymphocytes from Human Immunodeficiency Virus-Infected Individuals

    PubMed Central

    Paiardini, Mirko; Galati, Domenico; Cervasi, Barbara; Cannavo, Giuseppe; Galluzzi, Luca; Montroni, Maria; Guetard, Denise; Magnani, Mauro; Piedimonte, Giuseppe; Silvestri, Guido

    2001-01-01

    Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4+ T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G0 and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new

  13. 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

  14. Efficacy, durability, and response predictors of low-dose interleukin-2 therapy for chronic graft-versus-host disease.

    PubMed

    Koreth, John; Kim, Haesook T; Jones, Kyle T; Lange, Paulina B; Reynolds, Carol G; Chammas, Marie J; Dusenbury, Katherine; Whangbo, Jennifer; Nikiforow, Sarah; Alyea, Edwin P; Armand, Philippe; Cutler, Corey S; Ho, Vincent T; Chen, Yi-Bin; Avigan, David; Blazar, Bruce R; Antin, Joseph H; Ritz, Jerome; Soiffer, Robert J

    2016-07-01

    Chronic graft-versus-host disease (cGVHD) is associated with inadequate reconstitution of tolerogenic CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs). Previous phase 1 studies identified a low daily dose of interleukin-2 (IL-2) that was well tolerated, did not exacerbate alloimmunity, augmented Treg in vivo, and was associated with improvement of active cGVHD. In the current phase 2 study, 35 adults with steroid-refractory cGVHD received daily IL-2 (1 × 10(6) IU/m(2)) for 12 weeks. Median time from transplantation and cGVHD onset was 616 days (range, 270-2145 days) and 317 days (range, 28-1880 days), respectively. Two patients withdrew and 5 required IL-2 dose reductions due to side effects. Twenty of 33 evaluable patients (61%) had clinical responses at multiple cGVHD sites (liver, skin, gastrointestinal tract, lung, joint/muscle/fascia). Three patients (9%) had progressive cGVHD. Compared with pretreatment levels, Treg and natural killer cell counts rose >fivefold (P < .001) and >fourfold (P < .001), respectively, without significant change in conventional CD4 T cells (Tcons) or CD8 T cells. The Treg:Tcon ratio rose >fivefold (P < .001). Clinical responders initiated IL-2 earlier (508 vs 917 days after transplantation, P = .005; 249 vs 461 days after cGVHD onset; P = .03). Treg:Tcon ratios ≥0.07 at baseline and ≥0.2 at week 1 also predicted clinical response (P = .003; P = .0003, respectively). After a 4-week treatment hiatus, clinical responders were eligible to continue IL-2 therapy indefinitely. During 2 years of extended IL-2 therapy, clinical and Treg immune responses persisted, while Tcon count and Treg:Tcon ratio gradually normalized. Low-dose IL-2 provides durable clinical improvement in active cGVHD and extended therapy is well-tolerated. PMID:27073224

  15. Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury1

    PubMed Central

    Segel, Michael J; Aqeilan, Rami; Zilka, Keren; Lorberboum-Galski, Haya; Wallach-Dayan, Shulamit B; Conner, Michael W; Christensen, Thomas G; Breuer, Raphael

    2005-01-01

    The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE664Glu, an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE664Glu. However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis. PMID:16191100

  16. 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 HCMV—namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity—but 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

  17. Capacity of different cell types to stimulate cytotoxic T lymphocyte precursor cells in the presence of interleukin 2.

    PubMed

    Dröge, W; Moyers, C; Wehrmaker, A; Schmidt, H; Panknin, S; Männel, D; Falk, W

    1984-06-01

    Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis. PMID:6233360

  18. 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 2–4 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

  19. Thyroid function abnormalities associated with the chronic outpatient administration of recombinant interleukin-2 and recombinant interferon-alpha.

    PubMed

    Jacobs, E L; Clare-Salzler, M J; Chopra, I J; Figlin, R A

    1991-12-01

    We prospectively examined thyroid function during and following chronic, outpatient therapy with recombinant interleukin-2 (rIL-2) and Roferon-A (rIFN-alpha 2a). Twenty-two of 30 patients with advanced renal cell carcinoma treated on a phase II open pilot study of concomitant rIL-2 and rIFN-alpha 2a were included. Serum levels of thyroxine, triiodothyronine, free thyroxine index, thyrotropin, antithyroid antibodies, and thyrotropin (TSH) receptor binding antibodies were measured before therapy and after every other cycle. Selected patients underwent studies after every cycle and following completion of therapy. Twenty patients (91%) developed laboratory evidence of thyroid dysfunction, 11 (50%) developed hypothyroidism, five (23%) had a biphasic pattern, and four (18%) had hyperthyroidism. The incidence of thyroid dysfunction increased with increased number of treatment cycles. Transient hyperthyroidism was noted in six of the 11 patients studied after the first cycle and persisted after cycle three in only two patients. Hypothyroidism was not observed after cycle 1, but became increasingly frequent between cycles 2 (56%) and 6 (90%). Thyroid function normalized following therapy in nine of 12 patients tested. Antithyroid antibodies were identified pretherapy in five patients (23%) and de novo in none; TSH receptor binding antibodies were not detected. This study demonstrates a remarkably high frequency of reversible thyroid dysfunction in patients with advanced renal cell carcinoma treated with repeated cycles of rIL-2 plus rIFN-alpha 2a. We conclude that chronic therapy with rIL-2 and rIFN-alpha 2a produces thyroid dysfunction in virtually all patients most likely secondary to a nonspecific, nonautoimmune, toxic manifestation of prolonged treatment. IL-2 therapy may, therefore, produce thyroid dysfunction by more than one mechanism. PMID:1768679

  20. MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and -15 Receptors in T Cells.

    PubMed

    Mocsár, Gábor; Volkó, Julianna; Rönnlund, Daniel; Widengren, Jerker; Nagy, Péter; Szöllősi, János; Tóth, Katalin; Goldman, Carolyn K; Damjanovich, Sándor; Waldmann, Thomas A; Bodnár, Andrea; Vámosi, György

    2016-07-12

    MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Förster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2Rα/IL-15Rα. The mobility of not only MHC I but also that of IL-2Rα/IL-15Rα increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400-600 to 200-300 nm, whereas those of IL-2Rα/IL-15Rα hardly changed. MHC I and IL-2Rα/IL-15Rα colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1- and IL-2Rα/IL-15Rα-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins. PMID:27410738

  1. 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 Cγ 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.

  2. Elevated soluble CD8 antigen and soluble interleukin-2 receptors in the sera of patients with juvenile rheumatoid arthritis.

    PubMed

    Lipnick, R N; Sfikakis, P P; Klipple, G L; Tsokos, G C

    1993-07-01

    Activated T lymphocytes release various molecules including soluble CD8 (sCD8) antigen and soluble interleukin-2 receptor (sIL-2R). Elevated serum sCD8 antigen levels have been found in patients with viral infections, certain hematologic malignancies, and rheumatoid arthritis. On the other hand, elevated serum levels of sIL-2R have been found in various diseases including juvenile rheumatoid arthritis (JRA). We measured sCD8 antigen and sIL-2R levels using enzyme-linked immunosorbent assays in the sera of 49 afebrile patients with JRA (systemic 15, polyarticular 16, and pauciarticular 18) and 16 normal children. Disease activity was classified as mild, moderate, and severe. Sera from patients with severe JRA expressed statistically significant higher levels of both sCD8 and sIL-2R, whereas patients with mild disease had the lowest levels. There were no differences in the serum sCD8 and sIL-2R levels between the groups of patients with pauciarticular-, systemic-, and polyarticular-onset disease. Patients who were treated with prednisone had statistically nonsignificant higher serum levels of sCD8 and sIL-2R. A statistically significant positive correlation was found between sCD8 and sIL-2R levels, sCD8 levels and erythrocyte sedimentation rate (ESR), and sIL-2R levels and ESR. Our findings further suggest the presence of activated lymphocytes in patients with JRA and show that sCD8 antigen serum levels correlate with both serum levels of sIL-2R and ESR and thus may represent alternative indicators of disease activity. PMID:8513595

  3. 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

  4. Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes.

    PubMed Central

    Augustine, J A; Sutor, S L; Abraham, R T

    1991-01-01

    Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation. Images PMID:1652056

  5. Necrotizing enterocolitis as an adverse effect of recombinant interleukin-2 and Ch14.18 in maintenance therapy for high-risk neuroblastoma.

    PubMed

    Levy, Gabriel; Bonnevalle, Michel; Rocourt, Nathalie; Sudour, Hélène; Defachelles, Anne-Sophie

    2015-05-01

    Recombinant interleukin-2 is used with ch14.18/CHO to improve the cytotoxic activity of NK lymphocytes against neoplastic cells. The efficacy of this treatment is limited by its potential side effects. We report an unusual case of necrotizing enterocolitis associated with the administration of interleukin-2 and ch14.18/CHO in maintenance therapy for localized NMyc amplified neuroblastoma (NBL). This case highlights the potentially significant toxicity of this immunotherapy that is currently being tested in the high-risk NBL-1.5 protocol. Further, short-term, medium-term, and long-term follow-up in this patient population will be warranted to judge the potential benefit of this treatment versus the short-term, medium-term, and long-term side effects in a patient population with an outcome that is better than that of stage 4 NBL patients. PMID:25730142

  6. Interleukin-2 enhances the natural killer cell response to Herceptin-coated Her2/neu-positive breast cancer cells.

    PubMed

    Carson, W E; Parihar, R; Lindemann, M J; Personeni, N; Dierksheide, J; Meropol, N J; Baselga, J; Caligiuri, M A

    2001-10-01

    The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene. PMID:11592078

  7. 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).

  8. Blocking the interleukin 2 (IL2)-induced systemic autophagic syndrome promotes profound antitumor effects and limits toxicity.

    PubMed

    Lotze, Michael T; Buchser, William J; Liang, Xiaoyan

    2012-08-01

    Cancer is the leading cause of death in the United States in those dying under the age of 85. Although cancer is increasingly controlled as a chronic disease, true cures of patients with metastatic epithelial malignancies have rarely been obtained with currently available systemic therapies. For example, administration of high-dose recombinant interleukin 2 (IL2), enhancing cytolytic immune cell proliferation and delivery, promotes complete antitumor responses in < 10% of treated individuals. Means to reduce the toxicity, attributed to a cytokine storm and an associated "systemic autophagic syndrome" as well as enhance efficacy and increase the potential set of malignancies in which it is applied (currently patients with renal cancer and melanoma) would be of great interest. IL2 promotes both T-cell and NK cell induction of immune cell-mediated autophagy (iC-MA) in tumor targets. We have demonstrated that HMGB1 is detected at high levels in the serum of IL2-treated mice with translocation to the cytoplasm from the nucleus in the liver, consistent with HMGB1's release in response to stress, and ability to sustain autophagy. Limiting autophagy in mice with coadministration of chloroquine (CQ) diminishes serum levels of HMGB1, cytokines (IFNG and IL6 but not IL18), and autophagic flux, attenuating weight gain, enhancing DC, T-cell and NK cell numbers, and promoting long-term tumor control in a murine hepatic metastases model. Autophagy (programmed cell survival) is a metabolic process associated with promotion of late cancer growth. In tumor cell lines, CQ treatment limits ATP production through inhibition of oxidative phosphorylation and promotion of apoptosis. CQ increases autophagic vacuoles and LC3-II levels in tumor cells, associated with increased annexin V(+)/PI(-) cells, cleaved-PARP, cleaved-CASP3, and cytochrome c release from mitochondria. These observations, limiting toxicity and prolonging antitumor effects, with a combination of IL2 and autophagy

  9. 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

  10. 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 α 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

  11. Extended continuous infusion low-dose recombinant interleukin-2 in advanced cancer: prolonged immunomodulation without significant toxicity.

    PubMed

    Caligiuri, M A; Murray, C; Soiffer, R J; Klumpp, T R; Seiden, M; Cochran, K; Cameron, C; Ish, C; Buchanan, L; Perillo, D

    1991-12-01

    In previous clinical trials, recombinant interleukin-2 (rIL-2) has been infused at high doses over short periods of time to generate lymphokine-activated killer (LAK) cells in vivo. These trials have been limited by severe toxicities, and the immunologic effects of rIL-2 have been transient. The present study was designed to assess the toxicity and immunologic effects of prolonged administration of low doses of rIL-2. In this phase I study, patients with advanced cancer were scheduled to receive intravenous (IV) infusion of rIL-2 without interruption for 3 months in an outpatient setting. Twenty-one patients received rIL-2 at doses ranging from 0.5 x 10(5) to 6.0 x 10(5) U/m2/d. Treatment was extremely well tolerated, and no patient experienced grade 3 or grade 4 toxicity. The lowest dose level (0.5 x 10(5) U/m2/d) did not have demonstrable immunologic activity. At doses of 1.5 x 10(5) and 4.5 x 10(5) U/m2/d, rIL-2 infusion resulted in the specific expansion of natural-killer (NK) cells (sixfold and ninefold increases, respectively, at these two dose levels) without any changes in B cells, T cells, neutrophils, or monocytes. Grade 2 toxicity was observed at the dose of 6.0 x 10(5) U/m2/d, as three patients required interruption of therapy and two patients who completed therapy developed transient hypothyroidism. In patients with increased NK cells, enhancement of non-major histocompatibility complex (MHC)-restricted cytotoxicity and increased generation of LAK cells in vitro were also demonstrated. Therapy with low-dose rIL-2 can be given safely in an uninterrupted fashion for prolonged periods of time in an outpatient setting. This results in selective expansion of NK cells in vivo with minimal toxicity. Further investigation of this schedule for immunomodulation in vivo should be pursued in phase II studies of both malignant and immunodeficient disease states. PMID:1960552

  12. Soluble interleukin-2 receptor is a thyroid hormone-dependent early-response marker in the treatment of thyrotoxicosis.

    PubMed Central

    Smallridge, R C; Tsokos, G C; Burman, K D; Porter, L; Cranston, T; Sfikakis, P P; Solomon, B L

    1997-01-01

    Thyrotoxic patients exhibit increased levels of immune activation molecules (soluble interleukin-2 receptor [sIL-2R], intercellular adhesion molecule-1 [ICAM-1], and endothelial-leukocyte adhesion molecule-1 [ELAM-1]) in serum, although the clinical significance of these measurements remains unclear. In a randomized 4-week study, we have recently shown that in the treatment of hyperthyroidism, the combination of cholestyramine and methimazole (MMI) resulted in faster lowering of serum thyroid-hormone levels than did MMI alone. Stored serial serum samples from patients participating in this randomized treatment trial were analyzed for sIL-2R, soluble ICAM-1 (sICAM-1), and soluble ELAM-1 (sELAM-1). The levels of all three molecules were elevated in patients with hyperthyroidism. Although the levels of sICAM-1 and sELAM-1 remained elevated through the 4-week follow-up period in both groups of patients, the sIL-2R levels (normal levels, 1.0 to 4.2 ng/ml) decreased significantly in the 10 patients who received cholestyramine in addition to MMI (week 0, 14.2 +/- 1.5 ng/ml; week 2, 10.8 +/- 1.2 ng/ml; week 4, 8.9 +/- 1.5 ng/ml). In eight patients who received MMI alone, sIL-2R decreased less rapidly (week 0, 12.3 +/- 1.4 ng/ml; week 2, 12.3 +/- 1.3 ng/ml; week 4, 10.9 +/- 1.3 ng/ml). sICAM-1 and sELAM-1 were elevated at baseline but did not decrease during therapy. In the former group, free thyroxine and free triiodothyronine decreased faster. These data show that levels of sIL-2R in serum, but not those of sICAM-1 and sELAM-1, may be of clinical use in the early follow-up evaluation of medically treated patients. PMID:9302209

  13. 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).

  14. Recombinant Interleukin-2 in Patients Aged Younger Than 60 Years With Acute Myeloid Leukemia in First Complete Remission

    PubMed Central

    Kolitz, Jonathan E.; George, Stephen L.; Benson, Don M.; Maharry, Kati; Marcucci, Guido; Vij, Ravi; Powell, Bayard L.; Allen, Steven L.; DeAngelo, Daniel J.; Shea, Thomas C.; Stock, Wendy; Bakan, Courtney E.; Hars, Vera; Hoke, Eva; Bloomfield, Clara D.; Caligiuri, Michael A.; Larson, Richard A.

    2014-01-01

    BACKGROUND Recombinant interleukin-2 (rIL-2) induces cellular cytotoxicity against leukemia blasts. Patients with acute myeloid leukemia (AML) in first complete remission (CR) may harbor minimal residual disease that is susceptible to rIL-2–activated effector cells. METHODS In the Cancer and Leukemia Group B (CALGB) 19808 study, patients with AML in first CR were randomly assigned after all planned chemotherapy to receive a 90-day course of subcutaneously administered rIL-2 or no further therapy. The primary objective was to compare disease-free survival (DFS) between the 2 treatment arms. A total of 534 patients achieved a CR, 214 of whom were randomized. Six courses of low-dose daily rIL-2 were given for the expansion of cytotoxic effector cells, each followed by 3-day high-dose boluses given to trigger cytotoxicity against minimal residual disease. RESULTS On the protocol-specified intention-to-treat analysis, the hazards ratio for DFS was 0.75 (95% confidence interval, 0.52–1.09; P =.13); the 5-year DFS rate was 42% in the observation arm and 53% in the rIL-2 treatment arm. The hazards ratio for overall survival (OS) was 0.88 (95% confidence interval, 0.54–1.23; P =.34); the 5-year OS rate was 58% for the observation arm and 63% for the rIL-2 treatment arm. Twenty-five of the 107 patients randomized to treatment with rIL-2 either refused or were unable to initiate therapy and 30 patients did not complete their assigned therapy. However, significant toxicities were not commonly observed. The trial design did not anticipate the difficulties patients would encounter with protocol compliance. CONCLUSIONS The efficacy of immunotherapy with rIL-2 administered after intensive postremission treatment was not assessed as planned because of unexpected refusals by patients and/or their physicians to comply with protocol-directed therapy. Neither DFS nor OS was found to be significantly improved. PMID:24382782

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

    PubMed Central

    Kilpinen, S; Hurme, M

    1998-01-01

    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. Images Figure 3 PMID:9741337

  16. Phase I study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2.

    PubMed Central

    Kroesen, B. J.; Buter, J.; Sleijfer, D. T.; Janssen, R. A.; van der Graaf, W. T.; The, T. H.; de Leij, L.; Mulder, N. H.

    1994-01-01

    In a phase I trial the toxicity and immunomodulatory effects of combined treatment with intravenous (i.v.) bispecific monoclonal antibody BIS-1 and subcutaneous (s.c.) interleukin 2 (IL-2) was studied in renal cell cancer patients. BIS-1 combines a specificity against CD3 on T lymphocytes with a specificity against a 40 kDa pancarcinoma-associated antigen, EGP-2. Patients received BIS-1 F(ab')2 fragments intravenously at doses of 1, 3 and 5 micrograms kg-1 body weight during a concomitantly given standard s.c. IL-2 treatment. For each dose, four patients were treated with a 2 h BIS-1 infusion in the second and fourth week of IL-2 therapy. Acute BIS-1 F(ab')2-related toxicity with symptoms of chills, peripheral vasoconstriction and temporary dyspnoea was observed in 2/4 and 5/5 patients at the 3 and 5 micrograms kg-1 dose level respectively. The maximum tolerated dose (MTD) of BIS-1 F(ab')2 was 5 micrograms kg-1. Elevated plasma levels of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were detected at the MTD. Flow cytometric analysis showed a dose-dependent binding of BIS-1 F(ab')2 to circulating T lymphocytes. Peripheral blood mononuclear cells (PBMCs), isolated after treatment with 3 and 5 micrograms kg-1 BIS-1, showed increased specific cytolytic capacity against EGP-2+ tumour cells as tested in an ex vivo performed assay. Maximal killing capacity of the PBMCs, as assessed by adding excess BIS-1 to the assay, was shown to be decreased after BIS-1 infusion at 5 micrograms kg-1 BIS-1 F(ab')2. A BIS-1 F(ab')2 dose-dependent disappearance of circulating mononuclear cells from the peripheral blood was observed. Within the circulating CD3+ CD8+ lymphocyte population. LFA-1 alpha-bright and HLA-DR+ T-cell numbers decreased preferentially. It is concluded that i.v. BIS-1 F(ab')2, when combined with s.c. IL-2, has a MTD of 5 micrograms kg-1. The treatment endows the T lymphocytes with a specific anti-EGP-2-directed cytotoxic potential. PMID

  17. 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.

  18. Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

    PubMed Central

    Garrity, P A; Chen, D; Rothenberg, E V; Wold, B J

    1994-01-01

    Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using

  19. Potentiation of human natural killer cell cytotoxicity by Salmonella bacteria is an interferon- and interleukin-2-independent process that utilizes CD2 and CD18 structures in the effector phase.

    PubMed Central

    Tarkkanen, J; Saksela, E

    1991-01-01

    Incubation of large granular lymphocytes (LGL) with glutaraldehyde-fixed bacteria stimulated in the supernatant the production of interferon (IFN), which proved to be mainly IFN-gamma. Even though IFN-gamma was produced upon exposure of LGL to bacteria, anti-IFN-gamma antibodies failed to interfere with induction of cytotoxicity by bacterial contact. Anti-IFN-gamma receptor antibodies had no effect on the induction of activated killing by bacterial contact either. We also tested the effect of anti-IFN-alpha antibody, but it failed to interfere with induction of cytotoxicity by bacterial contact. No interleukin-2 (IL-2) was detected in the culture supernatant of bacterially activated LGL by the mouse HT2 cell assay, nor did we detect any IL-2 mRNA in bacterially activated LGL by Northern RNA blot assay. Neutralizing anti-IL-2 antiserum had no effect on the induction of activated killing by bacterial contact, and recombinant IL-4 did not interfere with the induction of activated killing. We then studied the membrane structures involved in bacterially activated killing. Anti-CD18 monoclonal antibody did not interfere with the induction phase of bacterially activated killing. However, both anti-CD18 and anti-CD2 antibodies inhibited the effector phase of bacterially activated killing. The effector pathways utilized by activated LGL depended on the mode of activation in that even though bacterially activated LGL were sometimes blocked by anti-CD2 monoclonal antibody, recombinant-IL-2-stimulated LGL were not. In conclusion, our present results suggest that there may be mediators other than exogenously secreted IFNs and IL-2 which are responsible for the induction of activated killing after bacterial contact. CD18 and CD2 structures were shown to be involved in the effector phase of bacterially activated killing. PMID:1713200

  20. Survival in renal cell carcinoma-a randomized evaluation of tamoxifen vs interleukin 2, alpha-interferon (leucocyte) and tamoxifen.

    PubMed Central

    Henriksson, R.; Nilsson, S.; Colleen, S.; Wersäll, P.; Helsing, M.; Zimmerman, R.; Engman, K.

    1998-01-01

    Metastatic renal cell carcinoma (RCC) has a poor prognosis. Conventional treatment strategies, including chemotherapy and hormonal therapy, have limited value. Although encouraging results have been achieved in terms of objective response using immunological manipulations, no conclusive studies yet exist with a controlled comparative evaluation of survival. Therefore, the present study was undertaken, which compared one of the present (and presumed best) treatments, interleukin 2/interferon-alpha (IL-2/IFN-alpha) and tamoxifen, with a control arm of tamoxifen only. Tamoxifen has been shown to potentiate in vivo anti-tumour activity of IL-2, and because of its non-toxic behaviour it was included in both groups. The study was open, randomized and included seven institutions in Sweden. The patients were stratified according to the different centres involved. An interim analysis was planned when a minimum of 100 patients were evaluable. The 128 patients finally included had a histologically documented metastatic RCC, with a life expectancy of more than 3 months, a performance status WHO 0-2 and no prior chemo- or immunotherapy. Informed consent was obtained from each patient. The patients randomized to the control arm (n = 63) received only tamoxifen 40 mg p.o. daily for at least 1 year or until progression. The patients (n = 65) randomized to biotherapy received subcutaneous recombinant IL-2, leucocyte IFN-alpha in a treatment cycle of 42 days, as well as tamoxifen p.o. In the absence of undue toxicity or disease progression, these patients received one additional treatment cycle of 42 days followed by maintenance treatment, consisting of 5 days therapy every 4 weeks, for 1 year, or until proven progression. Only two patients in the tamoxifen-only group received immunotherapy when the disease progressed, but without any beneficial effect. All patients received appropriate local treatment when indicated. The interim analysis demonstrated no survival advantage for

  1. Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

    SciTech Connect

    Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P. )

    1989-09-01

    Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb.

  2. 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, María; 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-γ, 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 60–70% 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 INFγ. 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

  3. mRNA transcript therapy.

    PubMed

    Weissman, Drew

    2015-02-01

    mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery. PMID:25359562

  4. 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.

  5. In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity.

    PubMed

    Heller, L; Pottinger, C; Jaroszeski, M J; Gilbert, R; Heller, R

    2000-12-01

    When cancer cells, including melanoma cells, are genetically altered to secrete cytokines, irradiated and injected into subjects, long-term antitumour immunity is induced. Optimally, existing melanomas induced to produce cytokines in vivo could stimulate this same immune response. Although in vivo electroporation enhances plasmid expression, electroporation of plasmids encoding granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-2 (IL2) into B16 mouse melanomas did not significantly alter tumour growth at the concentration tested. Electrochemotherapy, which causes short-term, complete regressions of treated tumour but no resistance to challenge, was combined with plasmid delivery. The combination treatment resulted in the induction of long-term immunity to recurrence and resistance to challenge in up to 25% of mice. PMID:11198480

  6. Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment.

    PubMed

    Barbé, Sofie; Van Mellaert, Lieve; Theys, Jan; Geukens, Nick; Lammertyn, Elke; Lambin, Philippe; Anné, Jozef

    2005-05-01

    The search for effective means of selectively delivering high therapeutic doses of anti-cancer agents to tumors has explored a variety of systems in the last decade. The ability of intravenously injected clostridial spores to infiltrate and thence selectively germinate in the hypoxic regions of solid tumors is exquisitely specific, making this system an interesting addition to the anti-cancer therapy arsenal. To increase the number of therapeutic proteins potentially useful for cancer treatment we have tested the possibility of Clostridium acetobutylicum to secrete rat interleukin-2 (rIL2). Therefore, rIL2 cDNA was placed under the control of the endo-beta-1,4-glucanase promoter and signal sequence of C. saccharobutylicum. Recombinant C. acetobutylicum containing the relevant construct secreted up to 800 microgl(-1) biologically active rIL2. The obtained yield should be sufficient to provoke in vivo effects. PMID:15869963

  7. Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor.

    PubMed

    Lorberboum-Galski, H; Kozak, R W; Waldmann, T A; Bailon, P; FitzGerald, D J; Pastan, I

    1988-12-15

    IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation. PMID:3143716

  8. CD45RA+ and CD45RO+ T cells differ in susceptibility to cyclosporin A mediated inhibition of interleukin-2 production.

    PubMed

    Schwinzer, R; Siefken, R

    1996-03-01

    Lymphocytes in different states of activation use different intracellular signalling pathways and may therefore differ in their susceptibility to immunosuppressive agents. In this study we examined the proliferation and production of interleukin-2 (IL-2) by unprimed/naive CD4+CD45RA+ T cells and previously activated/memory CD4+CD45RO+ T cells from human peripheral blood when stimulated in vitro in the presence of cyclosporin A (CsA). Further, the dependency of the IL-2 response on calcium (Ca2+) ions was analysed by the addition of the chelating agent EGTA. The CD4+CD45RO+ memory T cells were shown to be less susceptible to CsA and less dependent on the level of Ca+ ions than the naive CD4+CD45RA+ T cells. The subcellular mechanisms involved in this difference and the potential clinical implications are discussed. PMID:8762014

  9. 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

  10. 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

  11. Interleukin-2 and concanavalin A upregulate a cat2 isoform encoding a high affinity L-arginine transporter in feline lymphocytes.

    PubMed Central

    Stevens, B R; Tellier, M; Harvey, W; Feldman, D H; Bosworth, J

    2000-01-01

    The immunological responses of activated lymphocytes are associated with increased nitric oxide (NO) biosynthesis. Studies in the literature have primarily approached control of NO by focusing on the regulation of the nitric oxide synthase (NOS) isoforms. However, the present study approaches the control of NO synthesis by addressing the regulation of L-arginine availability to lymphocytes via regulation of membrane transport. The guanidino nitrogen of L-arginine is the sole biosynthetic precursor of NO. We investigated cytokine and mitogen regulation of membrane L-arginine transporters for the first time in feline cells. Feline peripheral blood mononuclear cells were treated with interleukin-2 and concanavalin A, then alternatively spliced isoforms of L-arginine transporters known in other species were probed by RT-PCR, using various oligonucleotide primers that hybridized to several regions in common with the isoforms. Both high affinity and low affinity isoforms are encoded by mRNAs arising from mutually exclusive alternative splicing of the primary transcript. A region of 123 bp was obtained that encoded an extracellular polypeptide loop of 41 amino acids. The sequence of this region represented the high affinity L-arginine substrate binding site of a CAT2 transporter polypeptide isoform, but not the CAT2a isoform low affinity binding site. Neither of the inducible isoforms were constitutively expressed in unstimulated feline cells. This is the first report demonstrating that domestic cats possess the cat2 gene encoding an inducible L-arginine transporter, and, furthermore, that the high affinity isoform transcript is activated by interleukin-2 and concanavalin A in feline lymphocytes. Images Figure 1. Figure 3. PMID:10935886

  12. Direct recognition of SLA- and HLA-like class II antigens on porcine endothelium by human T cells results in T cell activation and release of interleukin-2.

    PubMed

    Bravery, C A; Batten, P; Yacoub, M H; Rose, M L

    1995-11-15

    To investigate whether human T cells can directly recognize pig xenoantigens, highly purified human CD4+ and CD8+ T cells were incubated with pig aortic endothelial cells (PAEC). The response was measured by [3H]thymidine uptake and release of bioactive interleukin-2. A detailed examination of MHC expression by cultured PAEC and tissue sections of porcine aorta and heart showed porcine endothelial cells (EC) to be constitutively positive for SLA class II and antigens that crossreact with HLA class II molecules. Low level expression of B7 receptors was detected by binding of both human and mouse CTLA-4-Ig to untreated PAEC, which was enhanced significantly by treatment with recombinant porcine interferon-gamma. Human T cells, purified by positive selection and residual DR+ cells removed by lymphocytolysis, were shown to be functionally free of monocytes. Untreated PAEC elicited strong proliferation by human CD4+ T cells: CD8+ T cells also proliferated, but more weakly. This response was inhibited by CTLA-4-Ig. Blocking studies were performed with mAbs that bind to PAEC and not human EC (MSA3, TH16B), an mAb that binds to human and porcine EC (DA6.231), and L243, which binds to human and not porcine EC. The proliferative response of CD4+ T cells to PAEC was inhibited significantly by mAbs against swine and human determinants. In contrast, the response of CD4+ T cells to human EC was inhibited only by mAbs against human determinants. Experiments that directly compared the CD4+ and CD8+ T cell responses to PAEC and the human EC line EAhy.926, both with and without prior treatment with species-specific interferon gamma, demonstrated greater proliferation and 5-10 times more interleukin-2 in response to pig EC than to human EC. PMID:7491676

  13. Elimination of IgE regulatory rat CD8+ T cells in vivo differentially modulates interleukin-4 and interferon-gamma but not interleukin-2 production by splenic T cells.

    PubMed Central

    Diaz-Sanchez, D; Noble, A; Staynov, D Z; Lee, T H; Kemeny, D M

    1993-01-01

    Intraperitoneal immunization of Hooded Lister rats with a soluble antigen such as bee venom phospholipase A2 (PLA2), or ovalbumin (OVA) together with the toxic lectin, ricin, eliminates a population of early-activated CD8+ T cells which regulate IgE production. These early-activated CD8+ T cells are eliminated because they bear increased ricin-binding glycoproteins on their surface. This immunization regimen produces a vigorous and long-lived IgE response. The effect of this treatment on the capacity of splenic T cells to secrete interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and to generate IL-4 RNA message was assessed. IFN-gamma production by phytohaemagglutinin (PHA)- or ionomycin and phorbol myristate acetate (PMA)-stimulated splenocytes or purified splenic T cells from animals immunized with antigen and ricin was substantially reduced as compared with animals which were given saline or antigen alone (P < 0.001 Student's t-test). At the height of the primary IgE response IFN-gamma production by PHA-stimulated splenocytes was positively correlated with the number of CD8+ T cells (r = 0.90, P < 0.001) and inversely related to the level of serum IgE (r = -0.77, P < 0.020); serum IgE was inversely related to the number of CD8+ T cells (r = -0.92, P < 0.001). The reduced capacity of spleen cells from ricin and antigen immunized rats to produce IFN-gamma was first seen 7 days after immunization. The fall in the ability of splenocytes to secrete IFN-gamma closely paralleled the rise in serum IgE. IL-2 was assayed using an IL-2-dependent cell line which responded to rat IL-2 but not IL-4. Production of IL-2 by splenocytes taken from rats immunized with ricin+antigen was not significantly different to that produced by comparable cells obtained from animals immunized with antigen alone or saline. However, the levels of IL-4 mRNA, detected in ionomycin and PMA-stimulated splenocytes using a quantitative polymerase chain reaction (PCR) procedure, were three- to

  14. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    PubMed Central

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  15. 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

  16. [Prognostic predictive factors of the clinical response to immunotherapy with subcutaneous interleukin-2, in patients with metastatic renal carcinoma: analysis of 60 cases].

    PubMed

    Lissoni, P; Scardino, E; Favini, P; Barni, S; Tancini, G; Baccalin, A; Verweij, F; Strada, G; Musci, R; Rocco, F

    1995-04-01

    The intravenous immunotherapy with high-dose interleukin-2 (IL-2) would constitute one of the most effective treatments of metastatic renal cell carcinoma (RCC). More recently, IL-2 subcutaneous therapy has also appeared active, either alone or in association with interferon, with results comparable to those found with the intravenous route of injection, but with a lower toxicity. On this basis, we have designed a protocol of treatment with low-dose IL-2 alone given subcutaneously as a first or a second line therapy in metastatic RCC. The study included 60 consecutive patients (pts) (M/F: 39/21, median age 56 years, range 26/74). IL-2 was given at a dose of 3 millions IU twice/day for 5 days/week, for 6 weeks, corresponding to one cycle. In non progressed pts a second cycle was repeated after a 28-day rest period. Dominant metastasis sites were, as follows: soft tissues: 8; bone: 11; lung: 29; liver: 3; liver plus lung: 7; adrenal: 2. The minimum follow-up was 18 months and the median follow-up was 34 months (range 18-48). A complete response (CR) was achieved in 2/60 (3%) pts. A partial response (PR) was obtained in 15/60 (25%). Therefore, tumor objective rate (CR + PR) was 17/60 (28%). The median duration of response was 13 months (4-33).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7787857

  17. Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers

    PubMed Central

    Ito, Sawa; Bollard, Catherine M; Carlsten, Mattias; Melenhorst, Jan Joseph; Biancotto, Angélique; Wang, Ena; Chen, Jinguo; Kotliarov, Yuri; Cheung, Foo; Xie, Zhi; Marincola, Francesco; Tanimoto, Kazushi; Battiwalla, Minoo; Olnes, Matthew J; Perl, Shira; Schum, Paula; Hughes, Thomas E; Keyvanfar, Keyvan; Hensel, Nancy; Muranski, Pawel; Young, Neal S; Barrett, A John

    2014-01-01

    Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m2/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56bright NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors. PMID:24686272

  18. Effects of interleukin-2 therapy on the proliferation and differentiation of CD4/CD25 positive and CD4/CD25 negative cells in HIV+ patients.

    PubMed

    Caggiari, L; Zanussi, S; D'Andrea, M; Bortolin, M T; Crepaldi, C; Caffau, C; Paoli, P D

    2001-01-01

    Interleukin-2 has been widely used in HIV-1+ subjects as an immunoactivating agent. In this study, we investigated cytokine production, Ki67 antigen expression and the modulation of the surface phenotype of the CD4/CD25+ subset as compared to the reciprocal CD4/CD25- subset in IL-2-treated HIV+ patients. Our findings suggest that CD4 T cells are heterogeneous in responding to IL-2, because CD4/CD25+ cells sharply increased their "memory" phenotype, their Ki67 antigen expression and were the main in vivo targets for IL-2-dependent proliferation during therapy, while the percentages of IFN-gamma+ (terminally differentiated) cells remained unchanged at the end of therapy. Conversely, the CD4+/CD25- subpopulation showed an expansion of differentiated cells and a slight increase in the proliferation rate. The use of anti-retroviral therapy alone (HAART) reduced the proliferation and increased the differentiation of both CD4 subsets. Our data suggest that IL-2 has a moderate capacity to activate resting T cells in vivo and is probably unable to boost HIV-1 from latency to the replicative state. PMID:11566623

  19. 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 4α-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 IL15Rα+/− IgFc. PMID:25736261

  20. 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.

  1. Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin-2 on the release of acetylcholine in rat hippocampal slices

    PubMed Central

    Seto, David; Kar, Satyabrata; Quirion, Rémi

    1997-01-01

    The biphasic nature of the potent modulatory action of interleukin-2 (IL-2) on hippocampal acetylcholine (ACh) release was investigated by use of brain slice superfusion.Both the potentiating (10−13 M) and inhibitory (10−9 M) effects of IL-2 on hippocampal ACh release were stimulation-dependent and were blocked by a neutralizing IL-2 receptor antibody, suggesting the activation of typical IL-2 receptors in both cases.Tetrodotoxin (TTX; 10 μM) failed to block the potentiation of ACh release induced by a very low concentration of IL-2 (10−13M) suggesting a direct effect on cholinergic nerve terminals.In contrast, the inhibitory effect seen at a higher concentration (10−9 M) was TTX-sensitive, and hence indicative of an indirect action.To establish the nature of this intermediate mediator, blockers of nitric oxide synthesis, and of opioid and γ-aminobutyric acid (GABA) receptors were used. Only GABAA and GABAB receptor antagonists altered the inhibitory action of IL-2, suggesting the participation of GABA as mediator.Taken together, these results provide further evidence for the potent role of IL-2 in the modulation of cholinergic function in the rat hippocampus. PMID:9134229

  2. 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

  3. Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant.

    PubMed Central

    Bauer, K A; Ben-Bassat, A; Dawson, M; de la Puente, V T; Neway, J O

    1990-01-01

    A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth. PMID:2187412

  4. A phase II trial of concomitant human interleukin-2 and interferon-alpha-2a in patients with disseminated malignant melanoma.

    PubMed

    Whitehead, R P; Figlin, R; Citron, M L; Pfile, J; Moldawer, N; Patel, D; Jones, G; Levitt, D; Zeffren, J

    1993-02-01

    Interleukin-2 (IL-2) and alpha-interferon have each shown antitumor activity in patients with disseminated malignant melanoma. Because animal studies suggest enhanced activity for the combination over each agent used alone, this trial using a relatively low-dose outpatient regimen was undertaken. IL-2 at a dose of 2 x 10(6) U/m2/day (Roche units) was given by continuous intravenous infusion for 4 days a week with interferon-alpha-2a at a dose of 6 x 10(6) U/m2/day given by s.c. or i.m. injection on days 1 and 4 of each treatment week. One cycle consisted of 4 consecutive weeks of treatment followed by a 2-week rest period. Fourteen patients were entered in this study. No complete or partial responses were seen. One patient required dose reduction because of grade 3 diarrhea and two patients had interruption of treatment because of central-line-related sepsis. Fatigue was common in all patients. This low-dose combination regimen of IL-2 and alpha-interferon does not appear to be better than the single agents used alone in optimal dosage. PMID:8318496

  5. Aerosol Delivery of Interleukin-2 in Combination with Adoptive Transfer of Natural Killer Cells for the Treatment of Lung Metastasis: Methodology and Effect.

    PubMed

    Kiany, Simin; Gordon, Nancy

    2016-01-01

    Natural killer (NK) cells are a subtype of lymphocytes with a major role as a host defense mechanism against tumor cells. Allogeneic NK cell therapy is being used as an alternative promising therapy for many different cancers. Interleukin-2 (IL-2) is a critical cytokine for NK cell proliferation, survival, and effector functions. Cytokine support is essential to activate, expand, and increase the life span of NK cells. Aerosol delivery of IL-2 in combination with adoptive transfer of NK cells offers a reasonable approach for the treatment of lung metastases as it avoids the deleterious side effects of systemic IL-2. Using a human OS mouse model, we demonstrated the efficacy of this approach. Combination therapy of aerosol IL-2 with NK cells resulted in a better therapeutic effect against OS lung metastases as compared with each therapy alone. Aerosol IL-2 selectively increased infiltration, retention, and proliferation of infused NK cells in the lung, and there was no local inflammation or toxicity in the lungs or any other organ. Our results demonstrate that delivery of IL-2 via the aerosol route offers a feasible and innovative approach to enhance the immunotherapeutic effect of NK cells against pulmonary metastases. In the following chapter, we describe the methodology and effect of this innovative therapeutic approach. PMID:27177675

  6. Virus-Like Particles Displaying Recombinant Short-Chain Fragment Region and Interleukin 2 for Targeting Colon Cancer Tumors and Attracting Macrophages.

    PubMed

    Deo, Vipin Kumar; Kato, Tatsuya; Park, Enoch Y

    2016-05-01

    Functionalized virus-like particles (VLPs) can target with specificity as drug delivery systems and can attract macrophages for the destruction of cancer cells. Here, the group antigen capsid protein from the Rous sarcoma virus was used to prepare VLPs, functionalized by displaying glycol-inositol phosphate-anchored recombinant single chain fragment variable (rscFv) and hemagglutinin transmembrane region anchored recombinant human interleukin-2 (rhIL2) (designated as VLP-rscFv-rhIL2s) in silkworms. The rscFv specifically binds the tumor-associated glycoprotein 72 that is expressed at the surface of colon cancer cells. VLP-rscFv-rhIL2 was affinity purified and had a smooth particle size with a diameter of 50 nm. Calcein-AM-packaged VLP-rscFv-rhIL2s successfully targeted cancer cells as a model for drug delivery system. VLP-rscFv-rhIL2 bound with colon cancer cells that attracted macrophages (human monocytic cell line-1 cells) in chemotaxis chamber assays compared with negative controls. The macrophages secreted tumor necrosis factor-α, a cytokine that is necessary to destroy cancer cells. These results demonstrate the potential of VLP-rscFv-rhIL2 as an intelligent nano biomaterial that is capable of attracting macrophages. PMID:27037014

  7. Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: a prospective analysis.

    PubMed

    Brejcha, Martin; Stoklasová, Martina; Brychtová, Yvona; Panovská, Anna; Štěpanovská, Kristina; Vaňková, Gabriela; Plevová, Karla; Oltová, Alexandra; Horká, Kateřina; Pospíšilová, Šárka; Mayer, Jiří; Doubek, Michael

    2014-02-01

    Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p=0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period. PMID:24246692

  8. A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures.

    PubMed

    Sarkandy, Shahin Yegane; Khalilzadeh, Rasoul; Shojaosadati, Seyed Abbas; Sadeghizadeh, Majid; Farnoud, Amir Mohammad; Babaeipour, Valiollah; Maghsoudi, Amir

    2010-12-01

    A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 10³ to 8.08 × 10³ mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells. PMID:21062264

  9. Pulse interleukin-2 with famotidine induces CD56+ lymphocytes in the peripheral blood of patients with metastatic melanoma or kidney cancer.

    PubMed

    Quan, Walter D Y; Gagnon, Gregory A; Walker, Paul R; Quan, Francine M

    2011-02-01

    Increased lymphokine-activated killer (LAK) cell numbers and cytotoxicity against tumor cell lines have been seen in patients receiving high-dose continuous and bolus infusion interleukin-2 (IL-2) regimens. LAK are CD56 positive on flow cytometry. Daily intravenous doses of IL-2 of 18-21.6 MIU/m(2) over 15-30 minutes ("pulses") have been developed to attempt to lessen the toxicity of this therapy. It has been previously shown that the patients with metastatic melanoma or kidney cancer may be treated safely with pulse IL-2 daily for 5 days preceded by intravenous famotidine. Cycles were repeated every 21 days. Because LAK numbers have not been previously described with this regimen, the present study has examined CD56 numbers via peripheral blood flow cytometry in 11 patients with samples scheduled at baseline, after two cycles, and after four cycles. Eight (8) patients had melanoma and 3 had kidney cancer. Median CD56 counts after two cycles was significantly higher than baseline (p = 0.001). Similarly, CD56 counts at 2 months later were also greater than baseline (p = 0.009). There was no difference between median values after two cycles versus after four cycles. Patients who were clinical responders had a median CD56 count of 650 after two cycles when compared with nonresponders who had a median CD56 count of 290 (p = 0.005). CD56 counts are significantly elevated in patients treated with pulse IL-2 with famotidine and clinical responders have significantly higher CD56 than nonresponders. PMID:21348776

  10. A single nucleotide insertion in the canine interleukin-2 receptor gamma chain results in X-linked severe combined immunodeficiency disease.

    PubMed

    Somberg, R L; Pullen, R P; Casal, M L; Patterson, D F; Felsburg, P J; Henthorn, P S

    1995-08-01

    The immunologic and genetic analysis of a 14-week-old-male cardigan Welsh corgi puppy that presented with failure to thrive, diarrhea, and intermittent vomiting are described. The lack of palpable lymph nodes, the premature death of a male sibling, and similar clinical signs in a male cousin suggested that a primary immunodeficiency disease might be responsible for his poor clinical condition. Quantitation of serum immunoglobulins revealed low concentrations of IgG and undetectable IgA, yet normal concentrations of IgM. A complete blood cell count showed a slight anemia and lymphopenia. Although the peripheral blood contained a normal percentage of T cells, with an increased CD4:CD8 ratio, they were unable to proliferate in response to phytohemagglutinin (PHA) and/or interleukin 2 (IL-2). Furthermore, following PHA activation, the peripheral blood lymphocytes (PBL) demonstrated a nearly complete lack of IL-2 binding. All of these laboratory findings were identical with our previous findings from dogs with X-linked severe combined immunodeficiency (XSCID) that is due to a mutation in their IL-2 receptor gamma (IL-2R gamma) chain. Examination of the corgi's IL-2R gamma cDNA revealed an insertion of a cytosine following nucleotide 582, resulting in a premature stop codon prior to the transmembrane domain. The insertion also created an EcoO109 restriction enzyme site that enabled us to detect the mutation in the patient's genomic DNA. This new mutation in the IL-2R gamma chain discovered in a cardigan Welsh corgi puppy results in XSCID with similar immunologic abnormalities as observed in dogs with the same disease resulting from a different IL-2R gamma chain mutation. PMID:8571541

  11. Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

    PubMed Central

    Howell, M D; Diveley, J P; Lundeen, K A; Esty, A; Winters, S T; Carlo, D J; Brostoff, S W

    1991-01-01

    Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA. PMID:1660155

  12. Induction of interleukin 2 receptiveness and proliferation in resting peripheral T cells by monoclonal anti-CD3 (T3) antibodies does not require the presence of macrophages.

    PubMed Central

    Stingl, L A; Sinska, A; Landesmann, U; Smolen, J S

    1987-01-01

    In this study, we sought to elucidate the sequence of events by which mitogenic monoclonal anti-CD3 antibodies (anti-CD3-MoAb) initiate T cell activation. In cultures of monocyte-depleted resting T cells, two anti-CD3-MoAb, OKT3 and anti-Leu 4, induced a state of interleukin 2 (IL-2) receptiveness which culminated in T lymphocyte proliferation when recombinant IL-2 was provided. Evidence that Fc-receptor mediation by monocytes did not contribute to this mitogenesis was supported by studies showing that polyclonal F(ab')2 anti-mouse IgG Fc antibody did not alter the magnitude of the IL-2 driven T cell proliferative response, and by the use of T cells from donors whose monocytes were unable to assist in the induction of anti-Leu 4 (IgG1 subclass) initiated proliferation. Anti-CD3-MoAb, in the absence of IL-2, induced IL-2 receptor expression on purified T cells, and anti-IL 2 receptor antibodies inhibited T cell proliferation in the presence of this growth factor. Furthermore, following modulation of the CD3 molecular complex in the presence of monocytes, depletion of accessory cells rendered the modulated T cells mitogenically dependent on exogenous IL-2. IL-2 itself did not suffice to promote T cell proliferation in the absence of anti-CD3-MoAb. These results indicate that the binding of monoclonal antibody to CD3 is capable of initiating, in an accessory cell-independent manner, premitotic alterations in T cells which can culminate in proliferation when exogenous IL-2 is provided. PMID:3115639

  13. A Phase I Study of High-Dose Interleukin-2 With Sorafenib in Patients With Metastatic Renal Cell Carcinoma and Melanoma

    PubMed Central

    Lam, Elaine; Mortazavi, Amir; Kendra, Kari; Lesinski, Gregory B.; Mace, Thomas A.; Geyer, Susan; Carson, William E.; Tahiri, Sanaa; Bhinder, Arvinder; Clinton, Steven K.; Olencki, Thomas

    2014-01-01

    This study was designed to evaluate the safety and feasibility of high-dose interleukin-2 (HD IL-2) followed by sorafenib in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC). Biomarkers relevant to the antitumor effects of IL-2 that may be altered by sorafenib including the percentages of natural T-regulatory cells (Tregs), myeloid-derived suppressor cells (MDSC), and STAT5 phosphorylation (pSTAT5) in T cells were evaluated. We hypothesized that the proposed treatment schedule is feasible and safe and may lead to enhanced tumor response. A phase I dose escalation trial was conducted in patients with either metastatic RCC or MM. HD IL-2 (600,000 IU/kg IV q8h×8–12 doses) was administered on days 1–5 and 15–19, followed by sorafenib on days 29–82. The sorafenib dose was escalated. The percentage of Tregs, MDSC, and pSTAT5 in T cells were evaluated in peripheral blood by flow cytometry. Twelve of the 18 patients were evaluable for dose-limiting toxicity. No dose-limiting toxicity was observed. The treatment-related toxicity was predictable and did not seem to be additive with this schedule of administration. Partial responses were seen in 3 patients. No significant changes in the percentage of circulating Treg and MDSC were observed, whereas sorafenib did not adversely affect the ability of IL-2 to induce pSTAT5 in T cells. HD IL-2 followed by sorafenib was safe and feasible in patients with MM and RCC and did not adversely affect T-cell signaling through STAT5 in response to IL-2. PMID:24598448

  14. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP.

    PubMed Central

    Monfar, M; Lemon, K P; Grammer, T C; Cheatham, L; Chung, J; Vlahos, C J; Blenis, J

    1995-01-01

    Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C [cPKC])-dependent pp70S6k activation. The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k. Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity. Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k. In this case, inhibition appears to occur at least two points in this signalling path. Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K. The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain. PMID:7528328

  15. Relationship between Serum Level of Interleukin-2 in Patients with Systemic Lupus Erythematosus and Disease Activity in Comparison with Control Group

    PubMed Central

    Aghaei, Mehrdad; Musavi, Sara; Nomali, Mahin

    2014-01-01

    Background: Despite the large number of surveys, there are not any validated biomarkers for SLE disease activity till now. This study aimed to evaluate the relationship between serum level of IL-2 in patients with SLE and disease activity in comparison with control group. Materials and Methods: In this case-control study, 73 patients with lupus and 73 healthy subjects referred to the rheumatology clinic of 5 Azar Hospital in Gorgan (North of Iran).They were studied via convenience sampling during 2011-2012. Blood samples were taken from both groups and serum levels of interleukin -2 measured by Avi Bion Human IL-2 ELISA kit. Serum Level of IL-2 greater than 15 pg/ml defined positive and lesser than this amount defined negative. Disease activity evaluated with SLE disease activity index. Score greater than or equal to three or four defined as active disease. Data analysis conducted by SPSS software (version 16) and by using descriptive statistics and statistical tests. Results: Serum level of IL-2 was positive in 45.2% of sample studied and negative in 54.8% in case group, while in control group, serum level of IL-2 only in 11% of sample studied was positive and in 89% was negative. Statistical analysis indicated a significant relationship between serum level of IL-2 and the SLE disease activity index (p=0.025). Conclusion: This study showed the relationship between serum levels of IL-2 and disease activity, so this biomarker can be used as a clinical indicator for assessing disease activity in patients with SLE. PMID:25177590

  16. Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

    PubMed Central

    Rebollo, A; Silva, A

    1993-01-01

    In order to understand the respective roles of IL-2R alpha and IL-2R beta subunits in transmission of the interleukin-2 (IL-2)-mediated growth signals, we have established two IL-4-dependent murine T-cell clones stably expressing the human IL-2R beta chain and three clones stably expressing the human IL-2R alpha chain. Whereas parental LD8 cells (which express only the murine IL-2R beta chain) do not proliferate in response to IL-2, cell lines stably expressing human IL-2R beta or the chimeric IL-2R alpha beta complex proliferate in response to IL-2. Stably transfected cells expressing the chimeric high-affinity receptor (human IL-2R alpha and murine IL-2R beta) expressed de novo endogenous murine IL-2R alpha when cultured in the presence of IL-2 but not IL-4. Both chimeric and endogenous receptors are functional in response to IL-2, since only addition of both anti-human and anti-murine IL-2R alpha monoclonal antibodies (mAb) inhibited IL-2-induced proliferation. Taken together, our results strongly suggest that human and murine IL-2R beta molecules are different since interaction of IL-2 with human p70 IL-2R is sufficient for transduction of proliferative signals in the absence of p55 IL-2R or, alternatively, that over-expression of the IL-2R beta chain renders cells responsive to IL-2. In addition, IL-2 stimulation of T cells through different forms of IL-2R results in the induction of distinct cellular responses. PMID:8262551

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

    PubMed

    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

  18. Effects of phorbol esters and cytokines (interleukin-2,-4, and -6) on the proliferation and surface phenotype of Epstein-Barr virus immortalised human B lymphocytes.

    PubMed

    Kosmas, C; Epenetos, A A; Courtenay-Luck, N S

    1992-09-01

    Epstein-Barr virus (EBV)-induced in vitro infection of peripheral blood mononuclear cells (PBMCs) leads to a polyclonal proliferation and immortalisation of B lymphocytes. In the present study we determined the effects of three different cytokines, interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6), and the tumour promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on EBV-immortalised B lymphocytes. These factors have known activities on normal B cells. IL-4 and IL-6 increased significantly EBV-B cell proliferation after 3 and 5 days of culture, where IL-2 had no effect. The effect of IL-4 and IL-6 on EBV-B cells was abolished after pre-incubation with anti-IL-4 and anti-IL-6 neutralising antisera, respectively. TPA induced a dose dependent inhibition of proliferation both in serum free and 10% fetal calf serum (FCS) supplemented culture medium. Combinations of TPA and interleukins did not restore lymphoblastoid cell proliferation to background levels. All possible combinations of the three cytokines showed no synergistic or antagonistic effect on proliferation. TPA induced significant phenotypic changes of EBV immortalised B lymphocytes, by increasing IL-2 receptor (IL-2R) expression and decreasing CD20 and CD23 antigen expression. Other B cell differentiation antigens; HLA-DR, CD19, and transferrin receptor (CD71), did not demonstrate significant changes. A dose dependent inhibition of CD21 and increase in CD22 expression was observed in 2 out of 3 lymphoblastoid cell lines tested. PMID:1337296

  19. Retrospective Analysis of the Safety and Efficacy of High-dose Interleukin-2 After Prior Tyrosine Kinase Inhibitor Therapy in Patients With Advanced Renal Cell Carcinoma

    PubMed Central

    Wong, Michael K. K.; Agarwal, Neeraj; Redman, Bruce G.; Logan, Theodore; Gao, Dexiang; Flaig, Thomas W.; Lewis, Karl; Poust, Jamie; Monk, Paul; Jarkowski, Anthony; Sendilnathan, Arun; Bolden, Marcus; Kuzel, Timothy M.; Olencki, Thomas

    2014-01-01

    Although tyrosine kinase inhibitors (TKI) are the most common first-line therapy for metastatic renal cell carcinoma, high-dose interleukin-2 (HD-IL2) remains the only agent that provides durable complete responses. The optimal sequence of these agents remains uncertain. This retrospective multi-institutional study examined the safety and efficacy of HD-IL2 following TKI therapy. After IRB approval at 7 HD-IL2 centers, data relating to patient, disease, and treatment characteristics among 40 consecutive patients with metastatic renal cell carcinoma who were treated with HD-IL2 after at least 1 prior TKI therapy were retrospectively collected. The most common cardiac adverse events were grade 3 hypotension and vascular leak syndrome. Six patients (15%) experienced other grade ≥3 cardiac adverse events. There were 2 treatment-related deaths due to congestive heart failure, occurring in 1 patient with short TKI to HD-IL2 interval and another patient with an abnormal baseline cardiac stress test. Best responses included 2 CRs (5%, duration 40+ and 62+ mo), 3 PRs (8%, duration 6, 11, and 24 mo), 13 SD (32%, median duration 12 mo), 20 PD (50%), and 2 not evaluable patients. Median overall survival was 22 months. Administration of HD-IL2 could be safe and effective after TKI therapy; however, careful selection of patients is critical. We recommend baseline cardiac risk factor assessment, screening with both cardiac stress test and echocardiogram, and allowing a TKI to HD-IL2 interval of at least 2 months. PMID:25075565

  20. Retrospective analysis of the safety and efficacy of high-dose interleukin-2 after prior tyrosine kinase inhibitor therapy in patients with advanced renal cell carcinoma.

    PubMed

    Lam, Elaine T; Wong, Michael K K; Agarwal, Neeraj; Redman, Bruce G; Logan, Theodore; Gao, Dexiang; Flaig, Thomas W; Lewis, Karl; Poust, Jamie; Monk, Paul; Jarkowski, Anthony; Sendilnathan, Arun; Bolden, Marcus; Kuzel, Timothy M; Olencki, Thomas

    2014-09-01

    Although tyrosine kinase inhibitors (TKI) are the most common first-line therapy for metastatic renal cell carcinoma, high-dose interleukin-2 (HD-IL2) remains the only agent that provides durable complete responses. The optimal sequence of these agents remains uncertain. This retrospective multi-institutional study examined the safety and efficacy of HD-IL2 following TKI therapy. After IRB approval at 7 HD-IL2 centers, data relating to patient, disease, and treatment characteristics among 40 consecutive patients with metastatic renal cell carcinoma who were treated with HD-IL2 after at least 1 prior TKI therapy were retrospectively collected. The most common cardiac adverse events were grade 3 hypotension and vascular leak syndrome. Six patients (15%) experienced other grade ≥3 cardiac adverse events. There were 2 treatment-related deaths due to congestive heart failure, occurring in 1 patient with short TKI to HD-IL2 interval and another patient with an abnormal baseline cardiac stress test. Best responses included 2 CRs (5%, duration 40+ and 62+ mo), 3 PRs (8%, duration 6, 11, and 24 mo), 13 SD (32%, median duration 12 mo), 20 PD (50%), and 2 not evaluable patients. Median overall survival was 22 months. Administration of HD-IL2 could be safe and effective after TKI therapy; however, careful selection of patients is critical. We recommend baseline cardiac risk factor assessment, screening with both cardiac stress test and echocardiogram, and allowing a TKI to HD-IL2 interval of at least 2 months. PMID:25075565

  1. Francisella tularensis-induced in vitro gamma interferon, tumor necrosis factor alpha, and interleukin 2 responses appear within 2 weeks of tularemia vaccination in human beings.

    PubMed Central

    Karttunen, R; Surcel, H M; Andersson, G; Ekre, H P; Herva, E

    1991-01-01

    Cell-mediated immunity is essential for protection against the intracellular bacterium Francisella tularensis, which causes tularemia. Positive in vitro T-cell responses in the form of lymphocyte proliferation and lymphokine interleukin 2 (IL-2) and gamma interferon (IFN-gamma) secretion are found in memory immunity. Studies on the secretion of lymphokines with regard to the developing immunity to F. tularensis have not been published. Therefore, 14 subjects with no clinical history of tularemia were vaccinated with a live F. tularensis vaccine strain. The in vitro responses of five subjects (antigen-induced mononuclear cell and whole blood culture DNA synthesis and cytokine secretion) were measured twice a week throughout the period from 0 to 35 days after vaccination, and the peripheral blood lymphocyte subpopulations of nine subjects were determined between days 0 and 14. Positive reactions, i.e., responses exceeding those on day 0, were reached on day 10 with regard to the whole blood culture DNA synthesis response and IL-2 and IFN-gamma secretion and on day 14 with regard to the mononuclear cell DNA synthesis response and tumor necrosis factor alpha (TNF-alpha) secretion. No measurable IL-4 was found in either the immune or nonimmune supernatants. Since the secretion of TNF-alpha was related to immunization, this points to the specificity of the phenomenon, even though the type of secreting cell is not yet known. If it is shown later that specific T cells produce it, the TNF-alpha response and the negative IL-4 finding may speak for the importance of the Th1-like pattern in immunity to F. tularensis. PMID:1909711

  2. 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

  3. 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.

  4. Inability to Mediate Prolonged Reduction of Regulatory T Cells After Transfer of Autologous CD25-depleted PBMC and Interleukin-2 After Lymphodepleting Chemotherapy

    PubMed Central

    Powell, Daniel J.; de Vries, Christiaan R.; Allen, Tamika; Ahmadzadeh, Mojgan; Rosenberg, Steven A.

    2007-01-01

    Summary CD25+CD4+ regulatory T cells (Treg) regulate peripheral self-tolerance and possess the ability to suppress antitumor responses, which may explain the poor clinical response of cancer patients undergoing active immunization protocols, and provides the rationale for neutralizing Treg cells in vivo to strengthen local antitumor immune responses. Because interleukin-2 (IL-2) mediates tumor regression in about 15% of treated patients but simultaneously increases Treg cells, we hypothesized that transient elimination of Treg cells will enhance the clinical effectiveness of IL-2 therapy. In the current study, 5 patients with metastatic melanoma who were refractory to prior IL-2 received a lymphodepleting preparative regimen followed by the adoptive transfer of autologous lymphocytes depleted of CD25+ Treg cells and high-dose IL-2 administration. CD25+ cells were eliminated from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system, leaving CD8+ and CD25−CD4+ T cells intact. In the early aftermath of CD25+ Treg cell-depleted cell infusion, CD25+FOXP3+ CD4+ Treg cells rapidly repopulated the peripheral blood of treated patients with 18% to 63% of CD4+ T cells expressing FOXP3. Recovering CD25+CD4+ T cells exhibited suppressive activity against CD25−CD4+ effector T-cell proliferation in vitro. No patient experienced objective tumor regression or autoimmunity. Our results indicate that in vivo transfer of autologous CD25-depleted mononuclear populations to lymphopenic patients in combination with high-dose IL-2 is not sufficient to mediate prolonged reduction of Treg cells after IL-2 administration. PMID:17457218

  5. Spontaneous release of interleukin 2 by lung T lymphocytes in active pulmonary sarcoidosis is primarily from the Leu3+DR+ T cell subset.

    PubMed Central

    Saltini, C; Spurzem, J R; Lee, J J; Pinkston, P; Crystal, R G

    1986-01-01

    The inflammation within the lower respiratory tract of individuals with pulmonary sarcoidosis is dominated by large numbers of helper T lymphocytes that proliferate and spontaneously release interleukin 2 (IL-2). To identify the lymphocyte subpopulation that releases IL-2 in this disorder, lung lymphocytes recovered by bronchoalveolar lavage were characterized using the monoclonal antibodies Leu4 (T lymphocyte), Leu3 (helper/inducer), Leu2 (suppressor/cytotoxic), and anti-HLA-DR, and separated by panning and flow cytometry. The majority of the IL-2 spontaneously released by T cells in the sarcoid lung was contributed by the Leu3+ cell population (Leu3+65 +/- 23 IL-2 units released/10(6) cells per 24 h; Leu2+ 9 +/- 8, P less than 0.04). Further characterization of the lung Leu3+ T cells in sarcoid demonstrated that 30 +/- 3% were expressing HLA-DR molecules on their surface compared with 6 +/- 1% in normals (P less than 0.01). Importantly, the subpopulation of Leu3+ lung T lymphocytes expressing a high intensity of HLA-DR molecules on their surface was responsible for the majority of the release of IL-2 in the sarcoid lung (Leu3+ high-intensity DR 42 +/- 17 U/10(6) cells per 24 h, Leu3+ low-intensity DR 8 +/- 1 U/10(6) cells per 24 h; P less than 0.01). Thus, the spontaneous release of IL-2 in the lung of sarcoid patients appears to be localized to a subset of Leu3+ high-intensity DR ("activated" lung helper/inducer) T lymphocytes. Because the sarcoid lung is characterized by markedly increased numbers of these cells, it is likely that this compartmentalized T cell population plays a major role in sustaining the exaggerated localized immune processes of this disorder. PMID:3486888

  6. Effect of concanavalin A treatment on the allogeneic response of mice to challenge with P 815 mastocytoma: interleukin 2 treatment reverses concanavalin A suppression in vivo.

    PubMed

    Ekstedt, R D; Merdian, D J

    1984-05-01

    Mice injected repeatedly with concanavalin A (Con A) prior to and following challenge with P 815 mastocytoma are suppressed in their cell-mediated cytotoxicity responses. Earlier studies showed that pretreatment of the animals with silica to affect macrophage (M phi) functions reversed the Con A suppression. In the present paper we have shown that peritoneal exudate cells (PEC) induced/activated by ip injection of Con A were able to transfer suppression to normal mice. Separation of the PEC populations into adherent and nonadherent cells abrogated their capacity to transfer suppression. It was further shown that Con A is not functioning in this in vivo system to block effector activity of cytotoxic cells on target cells, and PEC induced with Con A were not directly cytotoxic to target P 815 cells. Finally, we were able to show that the cytotoxicity response of Con A-suppressed mice could be enhanced by treatment with concentrated culture supernatants of normal mouse spleen cells, rich in interleukin 2 (IL 2) activity. Attempts to detect a recently described mouse serum inhibitor of IL 2 in normal or Con A-treated mice were unsuccessful and spleen cells from Con A-treated mice lost their capacity to generate IL 2 in vitro when cultured under appropriate conditions. Taken together, these results suggest that suppression of cell-mediated immune responses in Con A-treated mice results from interruption of the normal generation of IL 2 helper effects necessary for the activation of cytotoxic effector T cells in vivo. PMID:6424952

  7. Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis

    PubMed Central

    McKinna, Lucy C.; Steinbach, Sabine; Dean, Gilly S.; Villarreal-Ramos, Bernardo; Whelan, Adam O.; Pirson, C.; Jones, Gareth J.; Clifford, Derek; Vordermeier, H. Martin

    2014-01-01

    We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed. PMID:24173026

  8. V delta 1 gene usage, interleukin-2 receptors and adhesion molecules on gamma delta+ T cells in inflammatory diseases of the nervous system.

    PubMed

    Mix, E; Fiszer, U; Olsson, T; Fredrikson, S; Kostulas, V; Söderström, M; Link, H

    1994-01-01

    This study investigates the expression of T cell receptor V delta 1 chain, interleukin-2 receptor alpha-chain (CD25) and adhesion molecules ICAM-1 (CD54), LFA-1 (CD11a/18) and CD44 on gamma delta+ T cells by three-color flow cytometry on cerebrospinal fluid (CSF) and blood cells in patients with multiple sclerosis (MS), other inflammatory neurological diseases (OIND) and other neurological diseases (OND). Of gamma delta + T cells in CSF and blood, 20-40% belonged to the 'epithelial' V delta 1 subtype. MS patients had the lowest levels in both CSF and blood, but the differences between the patient groups were not significant. The activation markers CD25 and CD54 were expressed by only a small proportion of gamma delta+ T cells and in a minority of patients. Although the occurrence of CD25+ and CD54+ gamma delta+ T cells was somewhat higher in CSF than in blood and in inflammatory diseases than in controls, the small numbers of CD25+ and CD54+ gamma delta+ T cells preclude establishing differences amongst compartments and patient groups. The adhesion molecules CD11a/18 and CD44 were constitutively expressed on all T cells. Therefore, we compared the relative antigen density per cell as measured by the relative fluorescence index (RFI) between CSF and blood, between the patient groups and between gamma delta+ and total T cells. The only difference encountered was a slightly higher expression of adhesion molecules on gamma delta+ compared to total T cells, with preference to MS patients. In conclusion, the V delta 1+ subtype of gamma delta+ T cells does not dominate in the CSF compartment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7507498

  9. 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

  10. Induction of synthesis of the cytolytic C9 (ninth component of complement)-related protein in human peripheral mononuclear cells by monoclonal antibody OKT3 or interleukin 2: correlation with cytotoxicity and lymphocyte phenotype

    SciTech Connect

    Martin, D.E.; Zalman, L.S.; Jung, G.; Mueller-Eberhard, H.J.

    1987-05-01

    Synthesis of the cytolytic C9-related protein (C9RP) was induced by activation of resting human peripheral T lymphocytes with the anti-CD3 antibody OKT3 or interleukin 2. Comparison of cellular cytotoxicity and C9RP content at various times during activation yielded a coefficient of correlation r = 0.92. During OKT3 stimulation of peripheral mononuclear cells, maximal C9RP content and cytotoxicity were observed by day 2 for 3, with subsequent decline to baseline values by day 5, whereas during interleukin 2 stimulation, both parameters reached the maximal level at days 3-5. After fluorescence-activated cell sorting, C9RP and cytotoxicity were quantitated in CD4/sup +/, CD8/sup +/, and Leu-19/sup +/ subsets. In OKT3-activated CD8/sup +/ cells, C9RP increased to approx. 3 x 10/sup 6/ molecules per cell, with a corresponding increase in lysis of human melanoma cells mediated by anti-CD3-anti-melanoma monoclonal antibody conjugates. Interleukin 2-stimulated CD8/sup +/ cell showed similar increases, but cytotoxicity was conjugate-independent. Activated CD4/sup +/ cells showed minimal increase in C9RP content. Leu-19/sup +/ cells, which exhibit natural killer cell activity, had a high C9RP content before stimulation.

  11. ROS activates JNK-mediated autophagy to counteract apoptosis in mouse mesenchymal stem cells in vitro

    PubMed Central

    Liu, Guan-yu; Jiang, Xiao-xue; Zhu, Xin; He, Wei-yang; Kuang, You-lin; Ren, Ke; Lin, Yong; Gou, Xin

    2015-01-01

    Aim: Transplantation of mesenchymal stem cells (MSCs) for the treatment of diabetic erectile dysfunction (ED) is hampered by apoptosis of the transplanted cells. In diabetic ED, there is increased oxidative stress and decreased NO in the corpora cavernosa, and reactive oxygen species (ROS) induce apoptosis of the transplanted cells. In this study we examined whether and how autophagy was involved in ROS-induced apoptosis of MSCs. Methods: Mouse C3H10 MSCs were treated with H2O2 to simulate the high oxidative condition in diabetic ED. Cell viability was measured using MTT assay. Apoptosis was analyzed by flow cytometry. Apoptosis- and autophagy-related proteins were detected with Western blot assays. Intracellular autophagosome accumulation was studied using transmission electron microscopy. Results: Treatment of MSCs with H2O2 (50–400 μmol/L) inhibited the cell viability in concentration- and time-dependent manners. Furthermore, H2O2 (300 μmol/L) induced apoptosis, as well as activated autophagy in MSCs. Pretreatment with lysosome inhibitor chloroquine (10 μmol/L) or PI3K inhibitor 3-methyladenine (5 mmol/L) significantly enhanced H2O2-induced cell death. Pretreatment with JNK inhibitor SP600125 (10 μmol/L) abrogated H2O2-induced accumulation of LC3-II, and attenuated H2O2-induced reduction of Bcl-2 levels in MSCs. Conclusion: ROS induce autophagy to counteract apoptosis in MSCs by activation of JNK. Thus, augmentation of autophagy may reduce apoptosis, prolonging MSC survival and improving MSC-based therapeutic efficacy for diabetic ED. PMID:26592514

  12. Acute and Chronic Hyperglycemia Elicit JIP1/JNK-Mediated Endothelial Vasodilator Dysfunction of Retinal Arterioles

    PubMed Central

    Hein, Travis W.; Xu, Wenjuan; Xu, Xin; Kuo, Lih

    2016-01-01

    Purpose Hyperglycemia, a hallmark of diabetes mellitus, is associated with retinal inflammation and impairment of endothelium-dependent nitric oxide (NO)–mediated dilation of retinal arterioles. However, molecular mechanisms involved in this diminished endothelial vasodilator function remain unclear. We examined whether inflammatory stress-activated kinases, c-Jun N-terminal kinase (JNK) and p38, contribute to retinal arteriolar dysfunction during exposure to acute and chronic hyperglycemia. Methods Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks; chronic hyperglycemia, 471 ± 23 mg/dL) or age-matched control pigs (euglycemia, 79 ± 5 mg/dL), and then cannulated and pressurized for vasoreactivity study. For acute hyperglycemia study, vessels from nondiabetic pigs were exposed intraluminally to high glucose (25 mM ≈ 450 mg/dL) for 2 hours, and normal glucose (5 mM ≈ 90 mg/dL) served as the control. Results Endothelium-dependent vasodilation to bradykinin was reduced in a similar manner after exposure to acute or chronic hyperglycemia. Administration of NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) nearly abolished vasodilations either in control (euglycemia and normal glucose) or hyperglycemic (acute and chronic) vessels. Treatment of either acute or chronic hyperglycemic vessels with JNK inhibitor SP600125 or JNK-interacting protein-1 (JIP1) inhibitor BI-78D3, but not p38 inhibitor SB203580, preserved bradykinin-induced dilation in an L-NAME–sensitive manner. By contrast, endothelium-independent vasodilation to sodium nitroprusside was unaffected by acute or chronic hyperglycemia. Conclusions Activation of JIP1/JNK signaling in retinal arterioles during exposure to acute or chronic hyperglycemia leads to selective impairment of endothelium-dependent NO-mediated dilation. Therapeutic targeting of the vascular JNK pathway may improve retinal endothelial vasodilator function during early diabetes. PMID:27556216

  13. Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis.

    PubMed

    Konishi, Hiroaki; Fujiya, Mikihiro; Tanaka, Hiroki; Ueno, Nobuhiro; Moriichi, Kentaro; Sasajima, Junpei; Ikuta, Katsuya; Akutsu, Hiroaki; Tanabe, Hiroki; Kohgo, Yutaka

    2016-01-01

    Previous reports have suggested that some probiotics inhibit tumorigenesis and cancer progression. However, the molecules involved have not yet been identified. Here, we show that the culture supernatant of Lactobacillus casei ATCC334 has a strong tumour-suppressive effect on colon cancer cells. Using mass spectrometry, we identify ferrichrome as a tumour-suppressive molecule produced by L. casei ATCC334. The tumour-suppressive effect of ferrichrome is greater than that of cisplatin and 5-fluorouracil, and ferrichrome has less of an effect on non-cancerous intestinal cells than either of those agents. A transcriptome analysis reveals that ferrichrome treatment induces apoptosis, which is mediated by the activation of c-jun N-terminal kinase (JNK). Western blotting indicates that the induction of apoptosis by ferrichrome is reduced by the inhibition of the JNK signalling pathway. This we demonstrate that probiotic-derived ferrichrome exerts a tumour-suppressive effect via the JNK signalling pathway. PMID:27507542

  14. Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis

    PubMed Central

    Konishi, Hiroaki; Fujiya, Mikihiro; Tanaka, Hiroki; Ueno, Nobuhiro; Moriichi, Kentaro; Sasajima, Junpei; Ikuta, Katsuya; Akutsu, Hiroaki; Tanabe, Hiroki; Kohgo, Yutaka

    2016-01-01

    Previous reports have suggested that some probiotics inhibit tumorigenesis and cancer progression. However, the molecules involved have not yet been identified. Here, we show that the culture supernatant of Lactobacillus casei ATCC334 has a strong tumour-suppressive effect on colon cancer cells. Using mass spectrometry, we identify ferrichrome as a tumour-suppressive molecule produced by L. casei ATCC334. The tumour-suppressive effect of ferrichrome is greater than that of cisplatin and 5-fluorouracil, and ferrichrome has less of an effect on non-cancerous intestinal cells than either of those agents. A transcriptome analysis reveals that ferrichrome treatment induces apoptosis, which is mediated by the activation of c-jun N-terminal kinase (JNK). Western blotting indicates that the induction of apoptosis by ferrichrome is reduced by the inhibition of the JNK signalling pathway. This we demonstrate that probiotic-derived ferrichrome exerts a tumour-suppressive effect via the JNK signalling pathway. PMID:27507542

  15. 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 ubiquitin–proteasome 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

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

    PubMed

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

    2015-02-01

    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

  17. 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

  18. 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

  19. Risk and outcome in metastatic malignant melanoma patients receiving DTIC, cisplatin, BCNU and tamoxifen followed by immunotherapy with interleukin 2 and interferon alpha2a.

    PubMed Central

    Hoffmann, R.; Müller, I.; Neuber, K.; Lassmann, S.; Buer, J.; Probst, M.; Oevermann, K.; Franzke, A.; Kirchner, H.; Ganser, A.; Atzpodien, J.

    1998-01-01

    Combined chemo-/immunotherapy has shown high objective response rates and a significant though small proportion of long-term complete responders in metastatic malignant melanoma. The purpose of this study was to determine response rates, freedom from treatment failure (FFTF) and overall survival in patients with advanced metastatic malignant melanoma treated with combined chemo-/immunotherapy, and to determine the value of a prognostic model for prediction of treatment outcome, FFTF and survival. Sixty-nine patients with metastatic malignant melanoma received combined chemo-/immunotherapy consisting of up to four cycles of DTIC (220 mg m(-2) i.v. days 1-3), cisplatin (35 mg m(-2) i.v. days 1-3), BCNU (150 mg m(-2) i.v. day 1, cycles 1 and 3 only) and tamoxifen (20 mg orally, daily). Two cycles of chemotherapy were followed by 6 weeks of outpatient immunotherapy with combined interleukin 2 (20 x 10(6) IU m(-2) days 3-5, weeks 1 and 4; 5 x 10(6) IU m(-2) days 1, 3, 5, weeks 2, 3, 5, 6) and interferon-alpha (6 x 10(6) IU m(-2) s.c. day 1, weeks 1 and 4; days 1, 3, 5, weeks 2, 3, 5, 6). All patients were evaluated on an intention-to-treat basis. Of 69 patients entered in the study, seven achieved complete remissions and 20 reached partial remissions with an objective response rate of 39% (95% confidence interval 28-52%). Median survival was 11 months, median FFTF was 5 months. Seven patients achieved ongoing long-term remissions, with maximum survival of 58 + months, and maximum FFTF of 58 + months. By Kaplan-Meier survival analysis and two-proportional Cox regression analysis, pretreatment performance status and serum lactic dehydrogenase were statistically significant and independent predictors of survival; risk groups could be defined as (a) the absence of both or (b) the presence of either one or both of these risk factors. Whereas survival and response were significantly influenced by patient risk, no influence could be demonstrated for FFTF. This combined

  20. Risk and outcome in metastatic malignant melanoma patients receiving DTIC, cisplatin, BCNU and tamoxifen followed by immunotherapy with interleukin 2 and interferon alpha2a.

    PubMed

    Hoffmann, R; Müller, I; Neuber, K; Lassmann, S; Buer, J; Probst, M; Oevermann, K; Franzke, A; Kirchner, H; Ganser, A; Atzpodien, J

    1998-10-01

    Combined chemo-/immunotherapy has shown high objective response rates and a significant though small proportion of long-term complete responders in metastatic malignant melanoma. The purpose of this study was to determine response rates, freedom from treatment failure (FFTF) and overall survival in patients with advanced metastatic malignant melanoma treated with combined chemo-/immunotherapy, and to determine the value of a prognostic model for prediction of treatment outcome, FFTF and survival. Sixty-nine patients with metastatic malignant melanoma received combined chemo-/immunotherapy consisting of up to four cycles of DTIC (220 mg m(-2) i.v. days 1-3), cisplatin (35 mg m(-2) i.v. days 1-3), BCNU (150 mg m(-2) i.v. day 1, cycles 1 and 3 only) and tamoxifen (20 mg orally, daily). Two cycles of chemotherapy were followed by 6 weeks of outpatient immunotherapy with combined interleukin 2 (20 x 10(6) IU m(-2) days 3-5, weeks 1 and 4; 5 x 10(6) IU m(-2) days 1, 3, 5, weeks 2, 3, 5, 6) and interferon-alpha (6 x 10(6) IU m(-2) s.c. day 1, weeks 1 and 4; days 1, 3, 5, weeks 2, 3, 5, 6). All patients were evaluated on an intention-to-treat basis. Of 69 patients entered in the study, seven achieved complete remissions and 20 reached partial remissions with an objective response rate of 39% (95% confidence interval 28-52%). Median survival was 11 months, median FFTF was 5 months. Seven patients achieved ongoing long-term remissions, with maximum survival of 58 + months, and maximum FFTF of 58 + months. By Kaplan-Meier survival analysis and two-proportional Cox regression analysis, pretreatment performance status and serum lactic dehydrogenase were statistically significant and independent predictors of survival; risk groups could be defined as (a) the absence of both or (b) the presence of either one or both of these risk factors. Whereas survival and response were significantly influenced by patient risk, no influence could be demonstrated for FFTF. This combined

  1. 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

  2. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-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 α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, 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-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα 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-2Rα 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-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  3. Durable responses and reversible toxicity of high-dose interleukin-2 treatment of melanoma and renal cancer in a Community Hospital Biotherapy Program

    PubMed Central

    2014-01-01

    Background High-dose interleukin-2 (IL-2) has been FDA-approved for over 20 years, but it is offered only at a small number of centers with expertise in its administration. We analyzed the outcomes of patients receiving high-dose IL-2 in relation to the severity of toxicity to ascertain if response or survival were adversely affected. Methods A retrospective analysis of the outcomes of 500 patients with metastatic renal cell carcinoma (RCC) (n = 186) or melanoma (n = 314) treated with high-dose IL-2 between 1997 and 2012 at Providence Cancer Center was performed. IL-2 was administered at a dose of 600,000 international units per kg by IV bolus every 8 hours for up to 14 doses. A second cycle was administered 16 days after the first and patients with tumor regression could receive additional cycles. Survival and anti-tumor response were analyzed by diagnosis, severity of toxicity, number of IL-2 cycles and subsequent therapy. Results The objective response rate in melanoma was 28% (complete 12% and partial 16%), and in RCC was 24% (complete 7% and partial 17%). The 1-, 2- and 3-year survivals were 59%, 41% and 31%, for melanoma and 75%, 56% and 44%, for RCC, respectively. The proportion of patients with complete or partial response in both melanoma and RCC was higher in patients who a) required higher phenylephrine doses to treat hypotension (p < 0.003), b) developed acidosis (bicarbonate < 19 mmol (p < 0.01)), or c) thrombocytopenia (<50, 50–100, >100,000 platelets; p < 0.025). The proportion achieving a complete or partial response was greater in patients with melanoma who received 5 or more compared with 4 or fewer IL-2 cycles (p < 0.0001). The incidence of death from IL-2 was less than 1% and was not higher in patients who required phenylephrine. Conclusions High-dose IL-2 can be administered safely; severe toxicity including hypotension is reversible and can be managed in a community hospital. The tumor response and survival

  4. 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

  5. A phase I study of prolonged continuous infusion of low dose recombinant interleukin-2 in melanoma and renal cell cancer. Part I: Clinical aspects.

    PubMed Central

    Vlasveld, L. T.; Rankin, E. M.; Hekman, A.; Rodenhuis, S.; Beijnen, J. H.; Hilton, A. M.; Dubbelman, A. C.; Vyth-Dreese, F. A.; Melief, C. J.

    1992-01-01

    The optimal schedule for recombinant interleukin-2 (rIL-2) administration is unclear. Because the clinical and immunological effects of prolonged continuous exposure to rIL-2 are unknown, we have conducted a phase I study to assess the toxicity and feasibility of continuous low dose infusion of rIL-2 (EuroCetus) using central venous access with a portable infusion device on an out-patient basis. Twenty-two patients entered the study, 13 with melanoma and nine with renal cell cancer, age range 26-66 years (median 51), performance status less than or equal to 1. They were treated with one of the following doses per m2 per 24 h: 0.18 x 10(6) IU, 0.6 x 10(6) IU, 1.8 x 10(6) IU, 3 x 10(6) IU, 6 x 10(6) IU and 9 x 10(6) IU. Toxicity was evaluable in 20 patients receiving greater than or equal to 3 weeks treatment duration or in whom treatment was discontinued prematurely because of toxicity. Constitutional symptoms consisting of fatigue, malaise and fever up to 40 degrees C without significant organ dysfunction occurred with doses greater than or equal to 1.8 x 10(6) IU m-2. The maximum tolerated dose was 6 x 10(6) IU m-2 24 h-1. In all patients toxicity reached a peak at 3 weeks and resolved thereafter despite continued rIL-2 treatment. Peripheral blood eosinophilia (up to 66% of white blood cell count) followed the same pattern. An infection of the central venous access occurred in 55% of the patients but this was mostly asymptomatic. Thirteen patients were treated greater than or equal to 6 weeks and were evaluable for tumour response. A partial remission occurred in a patient with melanoma with a dose of 1.8 x 10(6) IU rIL-2 m-2 24 h-1. PMID:1586602

  6. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-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 α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, 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-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα 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-2Rα 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-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  7. The role of interleukin-6 in mitogenic T-cell activation: detection of interleukin-2 heteronuclear RNA by polymerase chain reaction.

    PubMed

    Walz, G; Stevens, C; Zanker, B; Melton, L B; Clark, S C; Suthanthiran, M; Strom, T B

    1991-05-01

    It has been documented that interleukin-6 (IL-6) supports the proliferation of purified, anti-CD3-stimulated murine T cells. We found that stimulation of human peripheral blood mononuclear cells (PBMCs) with anti-CD3 induced a significant accumulation of IL-6 mRNA, indicating that antigen-mediated T-cell activation may involve IL-6 release from accessory cells. Phytohemagglutinin (PHA) had little effect upon IL-6 gene expression. In keeping with these findings, anti-IL-6 reduced but did not abolish anti-CD3-mediated proliferation of PBMCs, but had no significant effect upon PHA-stimulated proliferation. The addition of recombinant (r) IL-6 enhanced the proliferation of anti-CD3-stimulated PBMCs and increased the accumulation of IL-2 mRNA in PHA-stimulated PBMCs during the first 5 hr of culture. Nuclear run-off experiments did not reveal significant changes in IL-2 transcription in PHA plus rIL-6-treated PBMCs attempting to assume that IL-6 mediates stabilization of IL-2 mRNA. However, monitoring of partially spliced IL-2 mRNA by polymerase chain reaction revealed a clear increase in IL-2 heteronuclear RNA. Thus IL-6 increases the rate of IL-2 transcription which was not detectable by conventional in vitro transcription assays. We conclude that anti-CD3 triggers T-cell proliferation through a process that is partially but not entirely dependent upon release of IL-6. IL-6, in turn, supports IL-2 transcription. Insofar as anti-CD3 mimics antigen-triggered activation of the T-cell receptor complex, IL-6 appears to support the early immune response by augmenting antigen-triggered IL-2 gene expression. PMID:1827050

  8. Dynamics of the intracerebral and splenic cytokine mRNA production in Toxoplasma gondii-resistant and -susceptible congenic strains of mice.

    PubMed Central

    Deckert-Schlüter, M; Albrecht, S; Hof, H; Wiestler, O D; Schlüter, D

    1995-01-01

    Oral infection with a low-virulence strain of Toxoplasma gondii (Tg) induced a persisting encephalitis in resistant strains of mice. In the present study we have examined transcripts of various cytokines during acute and chronic stages of murine Tg encephalitis. In the brain of infected animals, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL-10 and IL-12 mRNA were induced to a significant extent, but only low levels of IL-4 mRNA were detectable. A similar cytokine profile was observed in the spleen. However, in contrast to the brain, the increase of IL-2 mRNA was particularly pronounced in the spleen, whereas the opposite was found for IFN-gamma and TNF-alpha mRNA. Thus, cytokines involved in T-cell proliferation were more prevalent in the spleen, but in the brain, where Tg actively multiplies, the effector molecules IFN-gamma and TNF-alpha were preferentially up-regulated. In addition, a detailed analysis of cytokine mRNA levels in major histocompatibility complex (MHC)-congenic strains of BALB and B10 mice revealed that the genetically regulated susceptibility to Tg was correlated with the amount of intracerebrally produced cytokine mRNA for IFN-gamma, TNF-alpha and IL-6. Mice with a strong increase of these cytokine mRNA were significantly better protected against Tg. This indicates that the outcome of toxoplasmosis may be critically dependent on an adequately regulated intracerebral immune response. Images Figure 1 Figure 2 Figure 3 PMID:7558129

  9. 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

  10. 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

  11. 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

  12. 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

  13. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

    PubMed

    Yang, Zhiyong; Edenberg, Howard J; Davis, Ronald L

    2005-01-01

    To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes. PMID:16204451

  14. 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.

  15. The cascading, interrelated roles of interleukin-1, interleukin-2, and interleukin-6 in murine anti-CD3-driven T cell proliferation.

    PubMed

    Pankewycz, O G; Yui, M; Kelley, V E; Strom, T B

    1990-04-01

    T cell stimulation occurs following interaction of T cell receptor (TcR) with processed antigen presented by autologous accessory cells (AC). The effects of antigen stimulation on T cells are mimicked by monoclonal antibodies (Mabs) defining proteins of the TcR-CD3 complex. In this study, we examine the roles of T cell density, AC density, divalent and polyvalent forms of anti-CD3 Mab, and the cytokines interleukin (IL)-1, IL-2, and IL-6 in T cell activation and proliferation. Stringently AC-depleted T cells do not proliferate in response to Con A or divalent anti-CD3; however, polyvalent anti-CD3 provides a powerful signal for isolated resting T cell proliferation. Recombinant (r)IL-2 strongly amplifies T cell proliferation in response to anti-CD3, whereas rIL-1 exerts no direct effects on anti-CD3-stimulated T cells. In the presence of AC, however, rIL-1 augments T cell proliferation to anti-CD3. Recombinant IL-6 promotes T cell proliferation among T cells stimulated with polyvalent but not divalent anti-CD3. As deduced by Northern blot analysis, rIL-1 increases cytoplasmic levels of IL-6 mRNA in AC. Recombinant IL-6, in turn, amplifies the accumulation of stable IL-2 transcripts in purified T cells stimulated with polyvalent anti-CD3. Hence, IL-1, IL-6, and IL-2 support T cell proliferation through cascading effects at the level of gene transcription. The cytokines evaluated in this study, however, do not fully reconstitute AC functions in promoting anti-CD3 Mab T cell proliferation. PMID:2137741

  16. 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

  17. 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

  18. Cytokine mRNA expression in lung tissue from toxic oil syndrome patients: a TH2 immunological mechanism.

    PubMed

    del Pozo, V; de Andrés, B; Gallardo, S; Cárdaba, B; de Arruda-Chaves, E; Cortegano, M I; Jurado, A; Palomino, P; Oliva, H; Aguilera, B; Posada, M; Lahoz, C

    1997-03-14

    In 1981, an epidemic occurred in Spain, toxic oil syndrome (TOS), in people who consumed rapeseed oil denatured with 2% aniline, and it was one of the largest intoxication epidemics ever recorded. In 1989, a similar disease, eosinophilia-myalgia syndrome (EMS) was reported in the USA and was associated with the ingestion of L-tryptophan. The pathologic findings in TOS showed primary endothelial injury, with cell proliferation and perivascular inflammatory infiltrates. Immunologic mechanisms have presumably been operative in the pathogenesis and perpetuation of TOS. Our previous findings pointed to a T-cell activation during acute phase of the disease. In order to analyze which T-cell subset is involved on TOS, we have developed an mRNA extraction procedure from paraffin-embedded lung tissues in patients with pulmonary involvement. We analyzed mRNA expression from different cytokines (IL-1, IL-2, IL-4, IL-5, IFN-gamma, GM-CSF) and CD25 (interleukin 2 receptor) and CD23 (low affinity IgE receptor), using RT-PCR technique. In lung tissues from these patients a T-cell activation was observed. We found a significant increase in Th1 (P = 0.006) and Th2 (P = 0.003) cytokine profile in TOS patients with respect to controls. The increment in TH2 response with respect to TH1 is significant (P = 0.03) in TOS lung specimens. Non-significant differences were obtained in other cytokines and receptors studied as IL-1, CD25, CD23 and GM-CSF. Data presented in this paper are the first clear evidence that an immunological mechanism is directly implicated in this illness. PMID:9074654

  19. 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.

  20. BAY 50-4798, a novel, high-affinity receptor-specific recombinant interleukin-2 analog, induces dose-dependent increases in CD25 expression and proliferation among unstimulated, human peripheral blood mononuclear cells in vitro.

    PubMed

    Matthews, Lynn; Chapman, Sherita; Ramchandani, Meena S; Lane, H Clifford; Davey, Richard T; Sereti, Irini

    2004-12-01

    Interleukin-2 administration induces CD4 T cell expansion in HIV-infected patients, however, toxicity can limit dosing. BAY 50-4798 is a recombinant IL-2 analog with >1000-fold specificity for the high-affinity IL-2 receptor. The effects of this compound on unstimulated human PBMC were evaluated. PBMC from HIV(-) and HIV(+) donors were cultured in vitro with incremental doses of BAY 50-4798 or aldesleukin. CD25 expression and proliferation were evaluated with flow cytometry. Cytokine levels were measured by ELISA in culture supernatants. BAY 50-4798 induced dose-dependent increases in CD25 expression and proliferation of T cells, NK, and B cells and showed selectivity for CD4 T cells expressing CD25. Induction of pro-inflammatory cytokines was also dose-dependent and was observed at the concentrations of BAY 50-4798 with the highest biologic activity. These data suggest that BAY 50-4798 can induce proliferation of unstimulated T cells but loss of T cell selectivity and induction of pro-inflammatory cytokines occur at concentrations exerting the highest biologic activity. PMID:15507389

  1. 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

  2. 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

  3. 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 1 week 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 8 months 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

  4. Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme.

    PubMed Central

    Schwer, B; Mao, X; Shuman, S

    1998-01-01

    Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action. Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure. Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs. ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature. Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1. These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability. The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated. PMID:9547258

  5. 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.

  6. mRNA quality control goes transcriptional

    PubMed Central

    Kilchert, Cornelia; Vasiljeva, Lidia

    2013-01-01

    Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5′ capped, spliced and 3′ polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC (quality control) mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs. Recently, with the growing appreciation that mRNA processing occurs concomitantly with polII (RNA polymerase II) transcription, it has become evident that QC acts at the transcriptional level in addition to degrading aberrant RNAs. In the present review, we discuss mechanisms that allow cells to co-transcriptionally initiate the removal of RNAs as well as down-regulate transcription of transcripts where processing repeatedly fails. PMID:24256272

  7. 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

  8. Interleukin-2 therapy for human cancer.

    PubMed

    van Hoesel, Q G

    1990-01-01

    The concept of immunotherapy is evolving from nonspecific, haphazard stimulation of the immune apparatus to more specific and controlled manipulation of the immune system. IL-2 gives the opportunity to exert influence on the cellular immune system. Why LAK cells are able to lyse tumor cells and leave normal cells intact is not known. How LAK cells behave after reinfusion is not known; are they able to migrate to tumor sites? Can improvements be made in scheduling in order to decrease toxicity and to enhance efficacy? But first of all, the question arises whether the tremendous efforts required by adoptive transfer, in terms of toxicity, logistics, and money, are outweighed by the therapeutic results. For clinical practice inside the frontier of oncology, continuous infusion of IL-2 at an intermediate dose is a quite attractive option in finding a balance between efforts and results. PMID:2117107

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

    PubMed

    Külshammer, 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

  10. Berberine Protects Human Umbilical Vein Endothelial Cells against LPS-Induced Apoptosis by Blocking JNK-Mediated Signaling

    PubMed Central

    Guo, Junping; Wang, Lijun; Wang, Linyao; Qian, Senmi; Fang, Jie

    2016-01-01

    Endothelial dysfunction is a critical factor during the initiation of atherosclerosis. Berberine has a beneficial effect on endothelial function; however, the underlying mechanisms remain unclear. In this study, we investigated the effects of berberine on lipopolysaccharide- (LPS-) induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the molecular mechanisms mediating the effect. The effects of berberine on LPS-induced cell apoptosis and viability were measured with 5-ethynyl-2′-deoxyuridine staining, flow cytometry, and Cell Counting Kit-8 assays. The expression and/or activation of proapoptotic and antiapoptotic proteins or signaling pathways, including caspase-3, poly(ADP-ribose) polymerase, myeloid cell leukemia-1 (MCL-1), p38 mitogen-activated protein kinase, C-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase, were determined with western blotting. The malondialdehyde levels, superoxide dismutase (SOD) activity, and production of proinflammatory cytokines were measured with enzyme-linked immunosorbent assays. The results demonstrated that berberine pretreatment protected HUVECs from LPS-induced apoptosis, attenuated LPS-induced injury, inhibited LPS-induced JNK phosphorylation, increased MCL-1 expression and SOD activity, and decreased proinflammatory cytokine production. The effects of berberine on LPS-treated HUVECs were prevented by SP600125, a JNK-specific inhibitor. Thus, berberine might be a potential candidate in the treatment of endothelial cell injury-related vascular diseases. PMID:27478481

  11. Inhibition of JNK-mediated autophagy enhances NSCLC cell sensitivity to mTORC1/2 inhibitors

    PubMed Central

    Jin, Hyeon-Ok; Hong, Sung-Eun; Park, Jin-Ah; Chang, Yoon Hwan; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    2016-01-01

    As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC. PMID:27358039

  12. Inhibition of JNK-mediated autophagy enhances NSCLC cell sensitivity to mTORC1/2 inhibitors.

    PubMed

    Jin, Hyeon-Ok; Hong, Sung-Eun; Park, Jin-Ah; Chang, Yoon Hwan; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    2016-01-01

    As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC. PMID:27358039

  13. Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells

    PubMed Central

    Li, Ting; Xu, Xiao-huang; Tang, Zheng-hai; Wang, Ya-fang; Leung, Chung-hang; Ma, Dik-lung; Chen, Xiu-ping; Wang, Yi-tao; Chen, Yi; Lu, Jin-jian

    2015-01-01

    Aim: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy. Methods: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg−1·d−1) was intraperitoneally injected to BEL-7402-bearing mice for 21 days. Results: Platycodin D (5–40 μmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 μmol/L at 24, 48 and 72 h, respectively. Platycodin D (5–20 μmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5–20 μmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 μmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg−1•d−1) significantly reduced relative tumor volume with decreased body weight. Conclusion: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors PMID:26592509

  14. 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

  15. 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

  16. 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

  17. Quercetin sensitizes pancreatic cancer cells to TRAIL-induced apoptosis through JNK-mediated cFLIP turnover.

    PubMed

    Kim, Ji Hye; Kim, Min Joo; Choi, Kyung-Chul; Son, Jaekyoung

    2016-09-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that can selectively kill cancer cells. Nonetheless, many cancers are resistant to TRAIL, and the molecular mechanisms of TRAIL resistance in cancer, particularly pancreatic cancer, are still unclear. In this study, we tested the hypothesis that quercetin, a flavonoid, induces apoptosis in TRAIL-resistant pancreatic cancer cells. Although quercetin alone had no significant cytotoxic effect, when combined with TRAIL, it promoted TRAIL-induced apoptosis that required mitochondrial outer membrane permeabilization. A BH3-only protein BID knockdown dramatically attenuated TRAIL/quercetin-induced apoptosis. The expression levels of cellular FLICE-like inhibitory protein (cFLIP) decreased in a dose-dependent manner in the presence of quercetin, and overexpression of cFLIP was able to robustly rescue pancreatic cancer cells from TRAIL/quercetin-induced apoptosis. Additionally, quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAIL-induced apoptosis. Thus, our results suggest that quercetin induces TRAIL-induced apoptosis via JNK activation-mediated cFLIP turnover. PMID:27477310

  18. A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells.

    PubMed

    Singh, Ajita V; Bandi, Madhavi; Raje, Noopur; Richardson, Paul; Palladino, Michael A; Chauhan, Dharminder; Anderson, Kenneth C

    2011-05-26

    Previous studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti-multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM. PMID:21454451

  19. Randomized study of intensified anthracycline doses for induction and recombinant interleukin-2 for maintenance in patients with acute myeloid leukemia age 50 to 70 years: results of the ALFA-9801 study.

    PubMed

    Pautas, Cecile; Merabet, Fatiha; Thomas, Xavier; Raffoux, Emmanuel; Gardin, Claude; Corm, Selim; Bourhis, Jean-Henri; Reman, Oumedaly; Turlure, Pascal; Contentin, Nathalie; de Revel, Thierry; Rousselot, Philippe; Preudhomme, Claude; Bordessoule, Dominique; Fenaux, Pierre; Terré, Christine; Michallet, Mauricette; Dombret, Hervé; Chevret, Sylvie; Castaigne, Sylvie

    2010-02-10

    PURPOSE In patients with acute myeloid leukemia (AML), induction chemotherapy is based on standard doses of anthracyclines and cytarabine. High doses of cytarabine have been reported as being too toxic for patients older than age 50 years, but few studies have evaluated intensified doses of anthracyclines. PATIENTS AND METHODS In this randomized Acute Leukemia French Association 9801 (ALFA-9801) study, high doses of daunorubicin (DNR; 80 mg/m(2)/d x 3 days) or idarubicin (IDA4; 12 mg/m(2)/d x 4 days) were compared with standard doses of idarubicin (IDA3; 12 mg/m(2)/d x 3 days) for remission induction in patients age 50 to 70 years, with an event-free survival (EFS) end point. After two consolidation courses based on intermediate doses of cytarabine, patients in continuous remission were randomly assigned to receive or not receive maintenance therapy with recombinant interleukin-2 (rIL-2; 5 x 10(6) U/m(2) x 5 days each month) for a total duration of 12 months. A total of 468 patients entered the study (median age, 60 years). Results Overall complete remission rate was 77% with significant differences among the three randomization arms (83%, 78%, and 70% in the IDA3, IDA4, and DNR arms, respectively; P = .04). However, no significant differences were observed in relapse incidence, EFS, or overall survival among the three arms. In the 161 patients randomly assigned for maintenance therapy, no difference in outcome was observed between the rIL-2 and the no further treatment arms. CONCLUSION Neither intensification of anthracycline doses nor maintenance with rIL-2 showed a significant impact on AML course, at least as scheduled in this trial. PMID:20048183

  20. Adjuvant treatment with interleukin-2- and interferon-alpha2a-based chemoimmunotherapy in renal cell carcinoma post tumour nephrectomy: Results of a prospectively randomised Trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN)

    PubMed Central

    Atzpodien, J; Schmitt, E; Gertenbach, U; Fornara, P; Heynemann, H; Maskow, A; Ecke, M; Wöltjen, H H; Jentsch, H; Wieland, W; Wandert, T; Reitz, M

    2005-01-01

    We conducted a prospectively randomised clinical trial to investigate the role of adjuvant outpatient immunochemotherapy administered postoperatively in high-risk patients with renal cell carcinoma. In total, 203 renal carcinoma patients' status post radical tumour nephrectomy were stratified into three risk groups: patients with tumour extending into renal vein/vena cava or invading beyond Gerota's fascia (pT3b/c pN0 or pT4pN0), patients with locoregional lymph node infiltration (pN+), and patients after complete resection of tumour relapse or solitary metastasis (R0). Patients were randomised to undergo either (A) 8 weeks of outpatient subcutaneous interleukin-2 (sc-rIL-2), subcutaneous interferon-alpha2a (sc-rIFN-α2a), and intravenous 5-fluorouracil (iv-5-FU) according to the standard Atzpodien regimen (Atzpodien et al, 2004) or (B) observation. Two-, 5-, and 8-year survival rates were 81, 58, and 58% in the treatment arm, and 91, 76, and 66% in the observation arm (log rank P=0.0278), with a median follow-up of 4.3 years. Two, 5-, and 8-year relapse-free survival rates were calculated at 54, 42, and 39% in the treatment arm, and at 62, 49, and 49% in the observation arm (log rank P=0.2398). Stage-adapted subanalyses revealed no survival advantages of treatment over observation, as well. Our results established that there was no relapse-free survival benefit and the overall survival was inferior with an adjuvant 8-week-outpatient sc-rIL-2/sc-rIFN-α2a/iv-5-FU-based immunochemotherapy compared to observation in high-risk renal cell carcinoma patients following radical tumour nephrectomy. PMID:15756254

  1. 1α,25-dihydroxyvitamin D3 in combination with transforming growth factor-β increases the frequency of Foxp3+ regulatory T cells through preferential expansion and usage of interleukin-2

    PubMed Central

    Chambers, Emma S; Suwannasaen, Duangchan; Mann, Elizabeth H; Urry, Zoe; Richards, David F; Lertmemongkolchai, Ganjana; Hawrylowicz, Catherine M

    2014-01-01

    A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune-mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3+ CD4+ T regulatory (Treg) cells when present at high concentrations or under strong T-cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3+ Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3+ Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10−7 m). Stimulation of human CD4+ T cells with a combination of 1,25(OH)2D3 and transforming growth factor-β (TGF-β) greatly increased the frequency of Foxp3+ Treg cells, which is proposed to result from the preferential expansion of Foxp3+ Treg cells, as compared with the Foxp3− effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin-2 by the Foxp3+ Treg cells compared with Foxp3− effector T cells. In summary, modulation of the cytokine environment to one high in TGF-β in the presence of 1,25(OH)2D3 (10−7 m) significantly increased Foxp3+ Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases. PMID:24673126

  2. Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

    PubMed Central

    Maass, G; Schmidt, W; Berger, M; Schilcher, F; Koszik, F; Schneeberger, A; Stingl, G; Birnstiel, M L; Schweighoffer, T

    1995-01-01

    Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7777545

  3. Interleukin-2 gene transfer potentiates the alpha-galactosylceramide-stimulated antitumor effect by the induction of TRAIL in NKT and NK cells in mouse models of subcutaneous and metastatic carcinoma.

    PubMed

    Nishihori, Yoshiki; Kato, Kazunori; Tanaka, Maki; Okamoto, Tetsuro; Hagiwara, Seiya; Araki, Naoko; Kogawa, Katsuhisa; Kuribayashi, Kageaki; Nakamura, Kiminori; Niitsu, Yoshiro

    2009-09-01

    Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects. PMID:19901518

  4. Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime-DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin-2-producing CD4 T-cell frequency post-vaccination.

    PubMed

    Kang, Han; Yuan, Qin; Ma, Hui; Hu, Zhi-Dong; Han, De-Ping; Wu, Kang; Lowrie, Douglas B; Fan, Xiao-Yong

    2014-12-01

    The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. Simple measurement of interferon-γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T-cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette-Guérin (BCG) prime-Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG-vaccinated mice, and the reduction in colony-forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin-2 (IL-2) -producing CD4 T cells and increased IL-2 production when measured as integrated mean fluorescence intensity post-vaccination as well. These data suggest that measurement of elevated frequency of IL-2-producing CD4 T cells or IL-2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime-boost strategies may be a useful approach to the control of M. tuberculosis infection. PMID:24965530

  5. Low-Dose Interleukin-2 Immunotherapy Does Not Improve Outcome of Patients Age 60 Years and Older With Acute Myeloid Leukemia in First Complete Remission: Cancer and Leukemia Group B Study 9720

    PubMed Central

    Baer, Maria R.; George, Stephen L.; Caligiuri, Michael A.; Sanford, Ben L.; Bothun, Sandra M.; Mrózek, Krzysztof; Kolitz, Jonathan E.; Powell, Bayard L.; Moore, Joseph O.; Stone, Richard M.; Anastasi, John; Bloomfield, Clara D.; Larson, Richard A.

    2008-01-01

    Purpose Cancer and Leukemia Group B (CALGB) 9720 evaluated subcutaneous low-dose recombinant interleukin-2 (rIL-2) maintenance immunotherapy as a strategy for prolonging remission in older patients with acute myeloid leukemia (AML). Patients and Methods AML patients age 60 years and older in first complete remission after induction and consolidation chemotherapy were randomly assigned to no further therapy or a 90-day regimen of 14-day cycles of low-dose rIL-2, aimed at expanding natural killer (NK) cells, followed by 3-day higher doses aimed at activating cytotoxicity of expanded NK cells to lyse residual AML cells. All randomly assigned patients were included in an intention-to-treat analysis. Results A total of 163 (64%) of 254 patients who completed induction and consolidation chemotherapy on CALGB 9720 were randomly assigned to rIL-2 (n = 81) or no further therapy (n = 82); the most common reasons for lack of random assignment were patient refusal and relapse. Fifteen patients randomly assigned to rIL-2 never initiated it because of refusal, intercurrent medical problems, or relapse, and 24 patients initiated rIL-2 but stopped early because of toxicity or relapse. Grade 4 toxicities during rIL-2 therapy included thrombocytopenia (65%) and neutropenia (64%), and grade 3 toxicities included anemia (33%), infection (24%) and malaise/fatigue (14%). Forty-two patients (52%) randomly assigned to rIL-2 completed the full 90-day course. Patients in both arms had similar distributions of both disease-free (combined median = 6.1 months; P = .47) and overall survival (combined median = 14.7 months; P = .61) after random assignment. Moreover, the 42 patients who completed all planned therapy did not show prolongation of disease-free or overall survival. Conclusion Low-dose rIL-2 maintenance immunotherapy is not a successful strategy in older AML patients. PMID:18591543

  6. Overproduction of human interleukin-2 in recombinant Escherichia coli BL21 high-cell-density culture by the determination and optimization of essential amino acids using a simple stoichiometric model.

    PubMed

    Yegane-Sarkandy, S; Farnoud, A M; Shojaosadati, S A; Khalilzadeh, R; Sadeghyzadeh, M; Ranjbar, B; Babaeipour, V

    2009-09-01

    In order to increase the productivity of human IL-2 (interleukin-2), a stoichiometric model has been used to determine the most essential amino acids and precise values of their amounts to be added to the culture during expression of human IL-2 (as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2). Experiments were performed to investigate the effect of chosen amino acids and their interactions on expression of human IL-2. Glutamine, a mixture of leucine, aspartic acid and glycine, and a mixture of leucine, glutamine and aspartic acid, were the most effective for the expression of IL-2. The most promising amino acids were then chosen for further experiments at three different levels to determine whether altering their stoichiometry can lead to better expression levels. The optimized value of glutamine in the flask was 0.316 g/l; a mixture of leucine, glutamine and aspartic acid at concentrations of 0.124, 0.316 and 0.212 g/l respectively and of leucine, aspartic acid and glycine in concentrations of 0.124, 0.212, 0.111 g/l respectively were chosen to be added to the flask. The effect of glutamine, as one of the amino acids most influencing the expression of IL-2 in batch and fed-batch high-cell-density cultures, was studied. The results revealed that the amount of expressed IL-2 compared with the control culture increased from 81 to 195 mg/l in the shake flask, 403 to 594 mg/l in the fermentor and 5.15 to 10.01 g/l in the fermentor under fed-batch cultivation. PMID:19341362

  7. Single mRNA Tracking in Live Cells

    PubMed Central

    Park, Hye Yoon; Buxbaum, Adina R.; Singer, Robert H.

    2011-01-01

    Asymmetric distribution of mRNA is a prevalent phenomenon observed in diverse cell types. The posttranscriptional movement and localization of mRNA provides an important mechanism to target certain proteins to specific cytoplasmic regions of their function. Recent technical advances have enabled real-time visualization of single mRNA molecules in living cells. Studies analyzing the motion of individual mRNAs have shed light on the complex RNA transport system. This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection. PMID:20580973

  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. Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer

    PubMed Central

    2014-01-01

    Background We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer. Patients and methods Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-α. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered. Results Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT. Conclusion AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer. PMID:24947606

  10. Seasonal changes in hepatic progesterone receptor mRNA, estrogen receptor mRNA, and vitellogenin mRNA in the painted turtle, Chrysemys picta.

    PubMed

    Custodia-Lora, Noemí; Callard, Ian P

    2002-10-01

    Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species. PMID:12392693

  11. Neo-adjuvant chemo-(immuno-)therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin + 5-fluorouracil (5-FU) with cisplatin + 5-FU + recombinant interleukin 2.

    PubMed

    Mantovani, G; Gebbia, V; Airoldi, M; Bumma, C; Contu, P; Bianchi, A; Ghiani, M; Dessì, D; Massa, E; Curreli, L; Lampis, B; Lai, P; Mulas, C; Testa, A; Proto, E; Cadeddu, G; Tore, G

    1998-11-01

    We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical response and toxicity of the combination chemotherapy cisplatin + 5-FU with the same combination plus s.c. recombinant interleukin-2 (rIL-2) in patients with advanced (stage III IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical Al Sarraf treatment: 100 mg/m2 cisplatin i.v. on day 1 plus 1000 mg m(-2) day(-1) 5-FU on days 1-5 as a continuous infusion. Regimen B was the same as regimen A plus 4.5 MIU/day rIL-2 s.c. on days 8-12 and 15-19. Treatment was repeated every 3 weeks for three cycles. A total of 33 patients were enrolled in the study; 30 were evaluable for toxicity and 28 for response. Seventeen patients were assigned to group A and 16 were assigned to group B. Three patients (20%) of group A and 4 (31%) of group B had a complete response, 9 patients (60%) of group A and 6 (46%) of group B had a partial response, with an overall response rate of 12 patients (80%) for group A and 10 patients (77%) for group B. Two patients (13%) of group A and 3 patients (23%) group B had stable disease; 1 patient (7%) of group A had progressive disease. Thus, there was not a statistically significant difference in response rate between the two groups and therefore there was no benefit from the addition of immunotherapy with rIL-2 to the standard chemotherapy. Both regimens were well tolerated. There were 2 toxic deaths (6.7%), 1 from hematological causes in group A and I from cardiac causes in group B. Myelosuppression and gastrointestinal toxicity, mainly nausea/vomiting and stomatitis, were the most frequent toxicities. The calculated number of patients for the sample has not yet been reached; however, the projection of our present results suggests that it is highly improbable that a clinically significant difference between the two treatment groups will be observed even if the calculated patient sample size is achieved

  12. Adjuvant low-dose interleukin-2 (IL-2) plus interferon-α (IFN-α) in operable renal cell carcinoma (RCC): a phase III, randomized, multicentre trial of the Italian Oncology Group for Clinical Research (GOIRC).

    PubMed

    Passalacqua, Rodolfo; Caminiti, Caterina; Buti, Sebastiano; Porta, Camillo; Camisa, Roberta; Braglia, Luca; Tomasello, Gianluca; Vaglio, Augusto; Labianca, Roberto; Rondini, Ermanno; Sabbatini, Roberto; Nastasi, Giuseppe; Artioli, Fabrizio; Prati, Andrea; Potenzoni, Michele; Pezzuolo, Debora; Oliva, Elena; Alberici, Federico; Buzio, Carlo

    2014-01-01

    There is currently no standard therapy to reduce the recurrence rate after surgery for renal cell carcinoma (RCC). The aim of this study was to assess efficacy and safety of adjuvant treatment with low doses of interleukin-2 (IL-2)+interferon-α (IFN-α) in operable RCC. The patients were randomized 1:1 to receive a 4-week cycle of low-dose IL-2+IFN-α or observation after primary surgery for RCC. Treatment cycles were repeated every 4 months for the first 2 years and every 6 months for the subsequent 3 years. The primary endpoint was recurrence-free survival (RFS); safety; and overall survival (OS) were secondary endpoints. ClinicalTrials.gov registration number was NCT00502034. 303/310 randomized patients (156 in the immunotherapy arm and 154 in the observation group) were evaluable at the intention-to-treat analyses. The 2 arms were well balanced. At a median follow-up of 52 months (range, 12-151 mo), RFS, and OS were similar, with an estimated hazard ratio (HR) of 0.84 [95% confidence interval (CI), 0.54-1.31; P=0.44] and of 1.07 (95% CI, 0.64-1.79; P=0.79), respectively in the 2 groups. Unplanned, subgroup analysis showed a positive effect of the treatment for patients with age 60 years and younger, pN0, tumor grades 1-2, and pT3a stage. Among patients with the combined presence of ≥ 2 of these factors, immunotherapy had a positive effect on RFS (HR=0.44; 95% CI, 0.24-0.82; P ≤ 0.01), whereas patients with <2 factors in the treatment arm exhibited a significant poorer OS (HR=2.27; 95% CI, 1.03-5.03 P=0.037). Toxicity of immunotherapy was mild and limited to World Health Organization grade 1-2 in most cases. Adjuvant immunotherapy with IL-2+IFN-α showed no RFS or OS improvement in RCC patients who underwent radical surgery. The results of subset analysis here presented are only hypothesis generating. PMID:25304727

  13. Here, there, everywhere. mRNA localization in budding yeast.

    PubMed

    Singer-Krüger, Birgit; Jansen, Ralf-Peter

    2014-01-01

    mRNA localization and localized translation is a common mechanism that contributes to cell polarity and cellular asymmetry. In metazoan, mRNA transport participates in embryonic axis determination and neuronal plasticity. Since the mRNA localization process and its molecular machinery are rather complex in higher eukaryotes, the unicellular yeast Saccharomyces cerevisiae has become an attractive model to study mRNA localization. Although the focus has so far been on the mechanism of ASH1 mRNA transport, it has become evident that mRNA localization also assists in protein sorting to organelles, as well as in polarity establishment and maintenance. A diversity of different pathways has been identified that targets mRNA to their destination site, ranging from motor protein-dependent trafficking of translationally silenced mRNAs to co-translational targeting, in which mRNAs hitch-hike to organelles on ribosomes during nascent polypeptide chain elongation. The presence of these diverse pathways in yeast allows a systemic analysis of the contribution of mRNA localization to the physiology of a cell. PMID:25482891

  14. 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

  15. Sodium Channel Inhibitors Reduce DMPK mRNA and Protein.

    PubMed

    Witherspoon, Luke; O'Reilly, Sean; Hadwen, Jeremiah; Tasnim, Nafisa; MacKenzie, Alex; Farooq, Faraz

    2015-08-01

    Myotonic dystrophy type 1 (DM1) is caused by an expanded trinucleotide (CTG)n tract in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This results in the aggregation of an expanded mRNA forming toxic intranuclear foci which sequester splicing factors. We believe down-regulation of DMPK mRNA represents a potential, and as yet unexplored, DM1 therapeutic avenue. Consequently, a computational screen for agents which down-regulate DMPK mRNA was undertaken, unexpectedly identifying the sodium channel blockers mexiletine, prilocaine, procainamide, and sparteine as effective suppressors of DMPK mRNA. Analysis of DMPK mRNA in C2C12 myoblasts following treatment with these agents revealed a reduction in the mRNA levels. In vivo analysis of CD1 mice also showed DMPK mRNA and protein down-regulation. The role of DMPK mRNA suppression in the documented efficacy of this class of compounds in DM1 is worthy of further investigation. PMID:26011798

  16. Probing dimensionality beyond the linear sequence of mRNA.

    PubMed

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions. PMID:26650615

  17. 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

  18. Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.

    PubMed

    Su, Wei; Slevin, Michael K; Marzluff, William F; Rhoads, Robert E

    2016-01-01

    Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3' stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5' → 3' and 3' → 5' processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5' and 3' termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5' → 3' degradation of a reporter mRNA containing canonical histone mRNA 3' SL and monitoring how oligouridylation and 3' → 5' degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3' SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5' or 3' termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing. PMID:27236794

  19. Role of T-helper type 2 cytokines in down-modulation of fas mRNA and receptor on the surface of activated CD4(+) T cells: molecular basis for the persistence of the allergic immune response.

    PubMed

    Spinozzi, F; Agea, E; Fizzotti, M; Bassotti, G; Russano, A; Droetto, S; Bistoni, O; Grignani, F; Bertotto, A

    1998-12-01

    The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation-induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL-2), IL-4, IL-5, transforming growth factor beta1, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro exposure of purified CD4(+) lymphocytes to allergen yielded only transient up-regulation of surface Fas but did not influence susceptibility to Fas-mediated cell death. T-helper type 2 cytokines (IL-4, IL-5, and GM-CSF) had a dose-dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure. PMID:9837865

  20. Production of granulocyte/macrophage-colony-stimulating factor by human natural killer cells. Modulation by the p75 subunit of the interleukin 2 receptor and by the CD2 receptor.

    PubMed

    Levitt, L J; Nagler, A; Lee, F; Abrams, J; Shatsky, M; Thompson, D

    1991-07-01

    Resting natural killer (NK) cells express the p75 chain of the IL-2 receptor (IL-2R beta) and most NK cells express the CD2 (erythrocyte rosette) receptor. The cell adhesion molecule, LFA-3, is a natural co-ligand for CD2. Tac antigen (IL-2R alpha), a p55 IL-2R subunit, can be expressed after NK activation and may play a role in IL-2-induced NK proliferation. Little is known of the molecular mechanisms underlying cytokine production in NK cells. We investigated the roles of IL-2R alpha, IL-2R beta, and CD2/LFA-3 in the molecular regulation of NK cell granulocyte/macrophage-colony-stimulating factor (GM-CSF) production. Enriched populations of peripheral blood NK cells were separated into CD16-positive and CD16-negative fractions by flow cytometry; positively selected cells were greater than 97% positive for CD16 (the FcIII receptor for IgG which is present on almost all NK cells), less than 1% positive for the T cell antigen CD3, and did not demonstrate rearrangement of the T cell receptor beta chain gene by Southern blot. NK cell supernatants were harvested after 3-4 d of incubation with 0-100 U/ml IL-2, or after incubation with anti-CD2 (T11(3] MAb and sheep red blood cells (SRBC are a homologue for LFA-3). Parallel cell aliquots were harvested at 3-16 h for transcriptional run-on assays, S1 nuclease assays, and actinomycin D mRNA t1/2 determinations. IL-2-activated NK supernatants contained large amounts of GM-CSF (178 +/- 35 pg/ml) by ELISA as did supernatants from CD2-activated NK cells (T11(3) MAb + SRBC: 212 +/- 42) vs. less than 20 pg/ml for NK cells incubated alone or with either SRBC or T11(3) MAb alone. Sepharose-linked anti-CD3 MAb did not induce GM-CSF release from NK cells. By S1 analysis, both IL-2 and CD2 stimulation markedly augmented GM-CSF mRNA expression but with very different latencies of onset. IL-2R beta MAb inhibited greater than 85% of GM-CSF release from IL-2-activated NK cells and markedly suppressed IL-2-induced GM-CSF mRNA expression

  1. Effects of DNA replication on mRNA noise.

    PubMed

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

    2015-12-29

    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

  2. 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

  3. Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

    PubMed Central

    Parra, E; Varga, M; Hedlund, G; Kalland, T; Dohlsten, M

    1997-01-01

    We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA

  4. 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

  5. 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

  6. mRNA redistribution during permanent focal cerebral ischemia.

    PubMed

    Lewis, Monique K; Jamison, Jill T; Dunbar, Joseph C; DeGracia, Donald J

    2013-12-01

    Translation arrest occurs in neurons following focal cerebral ischemia and is irreversible in penumbral neurons destined to die. Following global cerebral ischemia, mRNA is sequestered away from 40S ribosomal subunits as mRNA granules, precluding translation. Here, we investigated mRNA granule formation using fluorescence in situ histochemistry out to 8 h permanent focal cerebral ischemia using middle cerebral artery occlusion in Long Evans rats with and without diabetes. Neuronal mRNA granules colocalized with PABP, HuR, and NeuN, but not 40S or 60S ribosomal subunits, or organelle markers. The volume of brain with mRNA granule-containing neurons decreased exponentially with ischemia duration, and was zero after 8 h permanent focal cerebral ischemia or any duration of ischemia in diabetic rats. These results show that neuronal mRNA granule response has a limited range of insult intensity over which it is expressed. Identifying the limits of effective neuronal stress response to ischemia will be important for developing effective stroke therapies. PMID:24323415

  7. 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, 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. PMID:24904987

  8. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  9. Conventional and unconventional mechanisms for capping viral mRNA.

    PubMed

    Decroly, Etienne; Ferron, François; Lescar, Julien; Canard, Bruno

    2012-01-01

    In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5' ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5'-caps and escape innate immunity. PMID:22138959

  10. Effects of herpes simplex virus on mRNA stability.

    PubMed Central

    Strom, T; Frenkel, N

    1987-01-01

    Herpes simplex virus virions contain one or more functions which mediate shutoff of host protein synthesis, disaggregation of host polyribosomes, and degradation of host mRNA. We studied aspects of the host shutoff mechanism by using herpes simplex virus type 1 mutants deficient in virion-induced shutoff of host protein synthesis (G. S. Read and N. Frenkel, J. Virol. 46:498-512, 1983). Shutoff of host protein synthesis by the wild-type virus was associated with degradation of host mRNAs, including beta-actin, alpha-tubulin, and heat shock protein 70. In contrast, the virion host shutoff (vhs) mutants were deficient to various degrees in their ability to induce host mRNA degradation; the extent of mRNA degradation correlated well with the extent of inhibition of host protein synthesis. This finding suggests that inhibition of host protein synthesis and degradation of host mRNA were mediated by the same virion-associated function. Virion-induced degradation of host mRNA was not prevented by inhibitors of ribosome translocation, nor could it be augmented, for mutant vhs-1, by drugs which disaggregate polyribosomes. This suggests that mRNA in polyribosomes, as well as nonpolyribosomal mRNA, is susceptible to virion-induced degradation. Finally, the half-life of viral transcripts was also prolonged in cells infected with the vhs-1 mutant virus, suggesting that the vhs function indiscriminately decreased the half-lives of both host and viral mRNAs. The vhs function may thus play a dual role in virus infection. (i) It inhibits host gene expression, and (ii) it enables rapid transitions in the expression of viral genes which are sequentially transcribed as infection progresses. Images PMID:3035220

  11. mRNA Composition and Control of Bacterial Gene Expression

    PubMed Central

    Liang, S.-T.; Xu, Y.-C.; Dennis, P.; Bremer, H.

    2000-01-01

    The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-lacZ fusions (Pspc-lacZ, PRNAI-lacZ, and PRNAII-lacZ) was measured (i) in a relA+ background during exponential growth at different rates and (ii) in relA+ and ΔrelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp. PMID:10809680

  12. Rhythmic control of mRNA stability modulates circadian amplitude of mouse Period3 mRNA.

    PubMed

    Kim, Sung-Hoon; Lee, Kyung-Ha; Kim, Do-Yeon; Kwak, Eunyee; Kim, Seunghwan; Kim, Kyong-Tai

    2015-03-01

    The daily oscillations observed in most living organisms are endogenously generated with a period of 24 h, and the underlying structure of periodic oscillation is an autoregulatory transcription-translation feedback loop. The mechanisms of untranslated region (UTR)-mediated post-transcriptional regulation (e.g., mRNA degradation and internal ribosomal entry site (IRES)-mediated translation) have been suggested to fine-tune the expression of clock genes. Mouse Period3 (mPer3) is one of the paralogs of Period gene and its function is important in peripheral clocks and sleep physiology. mPer3 mRNA displays a circadian oscillation as well as a circadian phase-dependent stability, while the stability regulators still remain unknown. In this study, we identify three proteins - heterogeneous nuclear ribonucleoprotein (hnRNP) K, polypyrimidine tract-binding protein (PTB), and hnRNP D - that bind to mPer3 mRNA 3'-UTR. We show that hnRNP K is a stabilizer that increases the amplitude of circadian mPer3 mRNA oscillation and hnRNP D is a destabilizer that decreases it, while PTB exhibits no effect on mPer3 mRNA expression. Our experiments describe their cytoplasmic roles for the mRNA stability regulation and the circadian amplitude formation. Moreover, our mathematical model suggests a mechanism through which post-transcriptional mRNA stability modulation provides not only the flexibility of oscillation amplitude, but also the robustness of the period and the phase for circadian mPer3 expression. Mouse Period3 (mPer3) is one of well-known clock genes. We identified three 3'-UTR-binding proteins that modulate the mRNA stability, and they influenced to the amplitude of circadian mPer3 mRNA oscillation. Our mathematical model not only showed the relationship between mRNA stability and its oscillation profile but provided the molecular mechanism for the robustness of the period and the phase in circadian oscillation. hnK, heterogeneous nuclear ribonucleoprotein (hnRNP) K; hnD, hn

  13. 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

  14. SURVIV for survival analysis of mRNA isoform variation.

    PubMed

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  15. 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

  16. SURVIV for survival analysis of mRNA isoform variation

    PubMed Central

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  17. 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

  18. 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.

  19. Alternative ferritin mRNA translation via internal initiation

    PubMed Central

    Daba, Alina; Koromilas, Antonis E.; Pantopoulos, Kostas

    2012-01-01

    Ferritin stores and detoxifies an excess of intracellular iron, and thereby plays an important role in the metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival, and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. In light of the well-established transcriptional induction of ferritin by inflammatory signals, we examined how ferritin mRNA translation responds to stress conditions. We first used HT1080 fibrosarcoma cells engineered for coumermycin-inducible expression of PKR, a stress kinase that inhibits protein synthesis in virus-infected cells by phosphorylating eIF2α. In this setting, iron triggered partial ferritin mRNA translation despite a PKR-induced global shutdown in protein synthesis. Moreover, iron-mediated ferritin synthesis was evident in cells infected with an attenuated strain of poliovirus; when eIF4GI was cleaved, eIF2α was phosphorylated, and host protein synthesis was inhibited. Under global inhibition of protein synthesis or specific inhibition of ferritin mRNA translation in cells overexpressing PKR or IRP1, respectively, we demonstrate association of ferritin mRNA with heavy polysomes. Further experiments revealed that the 5′ untranslated region (5′ UTR) of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES). Our data are consistent with the existence of an alternative, noncanonical mechanism for ferritin mRNA translation, which may primarily operate under stress conditions to protect cells from oxidative stress. PMID:22271759

  20. 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.

  1. Influenza virus mRNA trafficking through host nuclear speckles.

    PubMed

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-01-01

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression. PMID:27572970

  2. 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

  3. Gene regulation by structured mRNA elements.

    PubMed

    Wachter, Andreas

    2014-05-01

    The precise temporal and spatial coordination of gene activity, based on the integration of internal and external signals, is crucial for the accurate functioning of all biological processes. Although the basic principles of gene expression were established some 60 years ago, recent research has revealed a surprising complexity in the control of gene activity. Many of these gene regulatory mechanisms occur at the level of the mRNA, including sophisticated gene control tasks mediated by structured mRNA elements. We now know that mRNA folds can serve as highly specific receptors for various types of molecules, as exemplified by metabolite-binding riboswitches, and interfere with pro- and eukaryotic gene expression at the level of transcription, translation, and RNA processing. Gene regulation by structured mRNA elements comprises versatile strategies including self-cleaving ribozymes, RNA-folding-mediated occlusion or presentation of cis-regulatory sequences, and sequestration of trans-acting factors including other RNAs and proteins. PMID:24780087

  4. Localization of histidine decarboxylase mRNA in rat brain.

    PubMed

    Bayliss, D A; Wang, Y M; Zahnow, C A; Joseph, D R; Millhorn, D E

    1990-08-01

    The recent cloning of a cDNA encoding fetal rat liver histidine decarboxylase (HDC), the synthesizing enzyme for histamine, allows the study of the central histaminergic system at the molecular level. To this end, Northern blot and in situ hybridization analyses were used to determine the regional and cellular distribution of neurons which express HDC mRNA in rat brain. Three hybridizing species which migrate as 1.6-, 2.6-, and 3.5-kb RNA were identified with Northern blots. The major (2.6 kb) and minor (3.5 kb) species, characteristic of HDC mRNA in fetal liver, were expressed at high levels in diencephalon and at just detectable levels in hippocampus, but not in other brain regions. In contrast, the 1.6-kb species was present in all brain regions examined except the olfactory bulb. Cells which contain HDC mRNA were found by in situ hybridization in the hypothalamus; HDC mRNA-containing cells were not detected in other areas, including the hippocampus. Hypothalamic neurons which express HDC mRNA were localized to all aspects of the tuberomammillary nucleus, a result consistent with previous immunohistochemical findings. PMID:19912749

  5. 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 ...

  6. Connections Underlying Translation and mRNA Stability.

    PubMed

    Radhakrishnan, Aditya; Green, Rachel

    2016-09-11

    Gene expression and regulation in organisms minimally depends on transcription by RNA polymerase and on the stability of the RNA product (for both coding and non-coding RNAs). For coding RNAs, gene expression is further influenced by the amount of translation by the ribosome and by the stability of the protein product. The stabilities of these two classes of RNA, non-coding and coding, vary considerably: tRNAs and rRNAs tend to be long lived while mRNAs tend to be more short lived. Even among mRNAs, however, there is a considerable range in stability (ranging from seconds to hours in bacteria and up to days in metazoans), suggesting a significant role for stability in the regulation of gene expression. Here, we review recent experiments from bacteria, yeast and metazoans indicating that the stability of most mRNAs is broadly impacted by the actions of ribosomes that translate them. Ribosomal recognition of defective mRNAs triggers "mRNA surveillance" pathways that target the mRNA for degradation [Shoemaker and Green (2012) ]. More generally, even the stability of perfectly functional mRNAs appears to be dictated by overall rates of translation by the ribosome [Herrick et al. (1990), Presnyak et al. (2015) ]. Given that mRNAs are synthesized for the purpose of being translated into proteins, it is reassuring that such intimate connections between mRNA and the ribosome can drive biological regulation. In closing, we consider the likelihood that these connections between protein synthesis and mRNA stability are widespread or whether other modes of regulation dominate the mRNA stability landscape in higher organisms. PMID:27261255

  7. Differential protein occupancy profiling of the mRNA transcriptome

    PubMed Central

    2014-01-01

    Background RNA-binding proteins (RBPs) mediate mRNA biogenesis, translation and decay. We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted cis-regulatory mRNA regions without a priori knowledge of the regulatory protein component. Results We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3′ UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells. Conclusions We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing cis-regulatory RNA sequence space and its dynamics in growth, development and disease. PMID:24417896

  8. Ochratoxin A mediates MAPK activation, modulates IL-2 and TNF-α mRNA expression and induces apoptosis by mitochondria-dependent and mitochondria-independent pathways in human H9 T cells.

    PubMed

    Darif, Youssef; Mountassif, Driss; Belkebir, Abdelkarim; Zaid, Younes; Basu, Kaustuv; Mourad, Walid; Oudghiri, Mounia

    2016-01-01

    Ochratoxin A (OTA) is a natural fungal secondary metabolite that contaminates food and animal feed. Human exposure and involvement of this mycotoxin in several pathologies have been demonstrated worldwide. We investigated OTA immunotoxicity on H9 cells, a human cutaneous CD4+ T lymphoma cell line. Cells were treated with 0, 1, 5, 10, and 20 µM OTA for up to 24 hr. Western blotting revealed increased phosphorylation of all three major mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun amino-terminal kinase, p38). OTA triggered mitochondrial transmembrane potential loss and caspase-3 activation. The 24-hr OTA treatment caused marked changes in cell morphology and DNA fragmentation, suggesting the occurrence of apoptotic events that involved a mitochondria-dependent pathway. Moreover, OTA triggered significant modulation of survivin, interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α): mRNA expression of survivin and IL-2 were decreased, while TNF-α was increased. OTA also caused caspase-8 activation in a time-dependent manner, which evokes the death receptor pathway activation; we suspect that this occurred via the autocrine pro-apoptotic effect of TNF-α on H9 cells. PMID:27193732

  9. Hfq affects mRNA levels independently of degradation

    PubMed Central

    2010-01-01

    Background The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. Results The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain. Conclusions The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome

  10. 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

  11. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    PubMed Central

    Thomsen, Rune; Daugaard, Tina F.; Holm, Ida E.; Nielsen, Anders Lade

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease. PMID:23991052

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

    PubMed Central

    Jerome, J F; Jaehning, J A

    1986-01-01

    We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708

  17. Leptin mRNA expresses in the bull reproductive organ.

    PubMed

    Abavisani, A; Baghbanzadeh, A; Shayan, P; Tajik, P; Dehghani, H; Mirtorabi, M

    2009-12-01

    Leptin, a 167-amino acid hormone, is secreted mainly by fat tissue. It has some powerful effects on the regulation of metabolism and reproductive function through endocrine and probably paracrine mechanisms. The contribution rate of leptin function on the male reproductive system is not still clear. Characterization of leptin expression in reproductive organs will suggest that in addition to its endocrine action, leptin has also paracrine/autocrine effects on reproduction. The expression of functional leptin receptor mRNA has been already recognized in testis of rodents, human and cattle. Thus, the aim of the present study was to investigate the presence of leptin mRNA in the bovine testis, because it will be the first step for understanding of its paracrine/autocrine effects on the male reproductive organs in cattle. The present study was the first to showed leptin mRNA expression in the testis of Holstein cattle using reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR products were amplified with nested PCR using inner leptin primer pairs to emphasis the first results. Besides, bovine beta actin gene was acted as an internal positive control as well as RNA purification marker. Our findings suggest that in addition to its endocrine actions at the hypothalamic-pituitary axis, leptin can has an autocrine and/or paracrine role in bull testicular function. PMID:19466574

  18. 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.

  19. Exaptive origins of regulated mRNA decay in eukaryotes.

    PubMed

    Hamid, Fursham M; Makeyev, Eugene V

    2016-09-01

    Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

  20. 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.

  1. Stochastic mRNA synthesis in mammalian cells.

    PubMed

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  2. Immunity of the Saccharomyces cerevisiae SSY5 mRNA to nonsense-mediated mRNA decay

    PubMed Central

    Obenoskey, Jesseeca; Lane, Dakota R.; Atkin, Audrey L.; Kebaara, Bessie W.

    2014-01-01

    The nonsense-mediated mRNA decay (NMD) pathway is a specialized pathway that triggers the rapid degradation of select mRNAs. Initially, identified as a pathway that degrades mRNAs with premature termination codons, NMD is now recognized as a pathway that also regulates some natural mRNAs. Since natural mRNAs do not typically contain premature termination codons, these mRNAs contain features that target them to NMD. In Saccharomyces cerevisiae mRNAs with atypically long 3′-UTRs are usually degraded by NMD, however in some conditions a constitutively expressed SSY5 mRNA with multiple NMD targeting signals including an atypically long 3′-UTR is an exception. We investigated the features of the SSY5 mRNAs that confer immunity to NMD. We found that the SSY5 mRNA 3′-UTRs are sufficient to target NMD insensitive mRNA to the pathway. Replacing the SSY5 3′-UTRs with the cyc1-512 3′-UTRs, known to target mRNAs to NMD or with the CYC1 3′-UTR, known not to target mRNAs to NMD, resulted in production of SSY5 mRNAs that were regulated by NMD. These observations suggest that the SSY5 mRNAs require sequences both within the 5′-UTR and/or ORF as well as the 3′-UTR to escape decay by NMD. PMID:25988166

  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. Body Fluid Identification Using mRNA Profiling.

    PubMed

    Roeder, Amy D; Haas, Cordula

    2016-01-01

    RNA analysis is a valuable tool for the identification of the forensically relevant body fluids, saliva, blood, menstrual blood, cervicovaginal fluid, and semen. Multiple human mRNA and bacterial RNA markers have been identified for each of these body fluids. RNA and DNA can be coextracted from the same portion of a sample and RNA markers for different body fluids can be multiplexed in a single PCR, thereby maximizing the number of analyses that can be performed with limited sample material. PMID:27259728

  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. Nucleolin mediates microRNA-directed CSF-1 mRNA deadenylation but increases translation of CSF-1 mRNA.

    PubMed

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K

    2013-06-01

    CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  7. Differential regulation of host mRNA translation during obligate pathogen-plant interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus infection reprograms the plant messenger RNA (mRNA) transcriptome by activating or interfering with a variety of signaling pathways, but the effects on host mRNA translation have not been explored on a genome-wide scale. To address this issue, Arabidopsis thaliana mRNA transcripts were quantif...

  8. RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

    PubMed Central

    Shen, V; Imamoto, F; Schlessinger, D

    1982-01-01

    Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro. Images PMID:6176575

  9. Subcellular mRNA localisation at a glance.

    PubMed

    Parton, Richard M; Davidson, Alexander; Davis, Ilan; Weil, Timothy T

    2014-05-15

    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

  10. Heterogeneity of zein mRNA and protein in maize.

    PubMed

    Park, W D; Lewis, E D; Rubenstein, I

    1980-01-01

    Zein, the prolamine fraction of maize, is localized in the endosperm in membrane-bound structures called protein bodies, which have polyribosomes on their surfaces. These polysomes or the mRNA fraction isolated from them will direct the synthesis of zein-like proteins in vitro. The in vitro products consist primarily of two molecular weight classes but show considerable charge heterogeneity when analyzed by isoelectric focusing. Although the molecular weight classes are very similar for different inbred lines, the isoelectric focusing patterns differ.Results given here suggest that the extensive charge heterogeneity of zein proteins does not result from the presence of a large number of totally distinct mRNAs. Zein proteins synthesized in vitro fall into several families related by sequence homologies in their mRNAs. In Illinois High Protein (IHP) the major zein mRNAs can be classified into three families based on their binding to cloned complimentary DNA copies of IHP zein mRNA. Each of three other lines we have studied (W22, Oh43, and W64A) has zein mRNAs that are related to those of IHP. Among these four lines the molecular weights of the members of a given family are generally similar, but the number of members in a family and their isoelectric points differ. PMID:16661153

  11. Sequence specificity of mRNA N6-adenosine methyltransferase.

    PubMed

    Csepany, T; Lin, A; Baldick, C J; Beemon, K

    1990-11-25

    The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells. PMID:2173695

  12. Motion of individual ribosomes along mRNA

    NASA Astrophysics Data System (ADS)

    Visscher, Koen

    2004-11-01

    Ribosomes move along messenger RNA to translate a sequence of ribonucleotides into a corresponding sequence of amino acids that make up a protein. Efficient motion of ribosomes along the mRNA requires hydrolysis of GTP, converting chemical energy into mechanical work, like better known molecular motors such as kinesin. However, motion is just one of the many tasks of the ribosome, whereas for kinesin, motion itself is the main goal. In keeping with these functional differences, the ribosome is also much larger consisting of more than 50 proteins and with half of its mass made up of ribosomal RNA. Such structural complexity enables indirect ways of coupling GTP hydrolysis to directed motion. In order to elucidate the mechanochemical coupling in ribosomes we have developed a single-molecule assay based on using optical tweezers to record the motion of individual ribosomes along mRNA. Translation rates of 2-4 codons/s have been observed. However, when increasing the force opposing motion, we observe backward slippage of ribosomes along homopolymeric poly(U) messages. Currently, it is not clear if the motor operates in reverse or if backward motion has become completely uncoupled from GTP hydrolysis. Interestingly, force-induced backward motion is of biological relevance because of its possible role in -1 frameshifting, a mechanism used by viruses to regulate gene expression at the level of translation.

  13. Tracking single mRNA molecules in live cells

    NASA Astrophysics Data System (ADS)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  14. Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.

    PubMed

    Benteyn, Daphné; Heirman, Carlo; Thielemans, Kris; Bonehill, Aude

    2016-01-01

    Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs. PMID:27236795

  15. Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins.

    PubMed

    Park, Mi Hee; Jo, Miran; Won, Dohee; Song, Ho Sueb; Song, Min Jong; Hong, Jin Tae

    2012-12-01

    We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals. PMID:23007278

  16. mTOR inactivation in myocardium from infant mice rapidly leads to dilated cardiomyopathy due to translation defects and p53/JNK-mediated apoptosis.

    PubMed

    Mazelin, Laetitia; Panthu, Baptiste; Nicot, Anne-Sophie; Belotti, Edwige; Tintignac, Lionel; Teixeira, Geoffrey; Zhang, Qing; Risson, Valérie; Baas, Dominique; Delaune, Emilie; Derumeaux, Geneviève; Taillandier, Daniel; Ohlmann, Théophile; Ovize, Michel; Gangloff, Yann-Gaël; Schaeffer, Laurent

    2016-08-01

    Mechanistic target of rapamycin (mTOR) is a central regulator of cell growth, proliferation, survival and metabolism, as part of mTOR complex 1 (mTORC1) and mTORC2. While partial inhibition of mTORC1 using rapamycin was shown to be cardioprotective, genetic studies in mouse models revealed that mTOR is essential for embryonic heart development and cardiac function in adults. However, the physiological role of mTOR during postnatal cardiac maturation is not fully elucidated. We have therefore generated a mouse model in which cardiac mTOR was inactivated at an early postnatal stage. Mutant mTORcmKO mice rapidly developed a dilated cardiomyopathy associated with cardiomyocyte growth defects, apoptosis and fibrosis, and died during their third week. Here, we show that reduced cardiomyocyte growth results from impaired protein translation efficiency through both 4E-BP1-dependent and -independent mechanisms. In addition, infant mTORcmKO hearts displayed markedly increased apoptosis linked to stretch-induced ANKRD1 (Ankyrin repeat-domain containing protein 1) up-regulation, JNK kinase activation and p53 accumulation. Pharmacological inhibition of p53 with pifithrin-α attenuated caspase-3 activation. Cardiomyocyte death did not result from activation of the MST1/Hippo pro-apoptotic pathway as reported in adult rictor/mTORC2 KO hearts. As well, mTORcmKO hearts showed a strong downregulation of myoglobin content, thereby leading to a hypoxic environment. Nevertheless, they lacked a HIF1α-mediated adaptive response, as mTOR is required for hypoxia-induced HIF-1α activation. Altogether, our results demonstrate that mTOR is critically required for cardiomyocyte growth, viability and oxygen supply in early postnatal myocardium and provide insight into the molecular mechanisms involved in apoptosis of mTOR-depleted cardiomyocytes. PMID:27133769

  17. 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

  18. Combination of Nimbolide and TNF-α-Increases Human Colon Adenocarcinoma Cell Death through JNK-mediated DR5 Up- regulation.

    PubMed

    Chantana, Chantana; Yenjai, Chavi; Reubroycharoen, Prasert; Waiwut, Pornthip

    2016-01-01

    Tumor necrosis factor (TNF-α), an inflammatory cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis, has previously been used in anti-cancer therapy. However, the therapeutic applications of TNF-α are largely limited due to its general toxicity and anti-apoptotic influence. To overcome this problem, the present study focused on the effect of active constituents isolated from a medicinal plant on TNF-α-induced apoptosis in human colon adenocarcinoma (HT-29) cells. Nimbolide from Azadirachta indica was evaluated for cytotoxicity by methyl tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and phase contrast microscopy. Effects on apoptotic signaling proteins were investigated using Western blot analysis. Nimbolide showed cytotoxicity against HT-29 cells that was significantly different from the control group (<0.01), a concentration of 10 μM significantly inducing cell death (<0.01). In combination with TNF-α, nimbolide significantly enhanced-induced cell death. In apoptotic pathway, nimbolide activated c-Jun N-terminal kinase (JNK) phosphorylation, BH3 interacting-domain death agonist (Bid) and up-regulated the death receptor 5 (DR5) level. In the combination group, nimbolide markedly sensitized TNF-α-induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance TNF-α-mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway. PMID:27268643

  19. Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins.

    PubMed

    Park, Mi Hee; Kim, Jong Han; Chung, Young-Ho; Lee, Seung Ho

    2016-04-29

    We investigated whether bakuchiol, an analog of resveratrol enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. Bakuchiol enhanced expression of cell death receptor (DR) in TRAIL-sensitive and -resistant colon cancer cells in a dose-dependent manner. A combination of bakuchiol with TRAIL significantly inhibited cell growth of TRAIL sensitive HCT116 and TRAIL resistant HT-29 cells. The expression of TRAIL receptors; DR4 and DR5 was significantly increased by treatment of bakuchiol, however, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed. Moreover, the expression of apoptosis related proteins such as cleaved caspase-3, -8, -9 and PARP was increased by combination treatment of bakuchiol and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory effects of bakuchiol in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the bakuchiol induced cell growth inhibitory effects. The collective results suggest that bakuchiol facilitates TRAIL-induced apoptosis in colon cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals. PMID:27033605

  20. Prodynorphin, proenkephalin and kappa opioid receptor mRNA responses to acute "binge" cocaine.

    PubMed

    Spangler, R; Zhou, Y; Maggos, C E; Schlussman, S D; Ho, A; Kreek, M J

    1997-02-01

    Previous studies showed that preprodynorphin (ppDyn) mRNA increases in caudate-putamen while kappa opioid receptor (KOR) mRNA decreases in substantia nigra after 3 and 14 days "binge" cocaine. To further characterize opioid mRNA responses, rats were administered: saline; 1 day cocaine followed by 1 day saline; 1 day cocaine; or 2 days cocaine. ppDyn mRNA in caudate-putamen increased in both groups receiving cocaine on the final day compared to groups receiving saline. Preproenkephalin (ppEnk) mRNA in caudate-putamen increased, and KOR mRNA in substantia nigra decreased, after 2 days of cocaine. Thus ppDyn mRNA is elevated acutely by cocaine, while ppEnk and KOR mRNAs show a significant response only on the second day of "binge" cocaine. PMID:9030708

  1. Identification of proteins specifically interacting with YB-1 mRNA 3' UTR and the effect of hnRNP Q on YB-1 mRNA translation.

    PubMed

    Lyabin, D N; Nigmatullina, L F; Doronin, A N; Eliseeva, I A; Ovchinnikov, L P

    2013-06-01

    In this study, proteins specifically interacting with the 3' untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3' UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator - YB-1 itself. PMID:23980891

  2. mRNA retroposition in human cells: processed pseudogene formation.

    PubMed Central

    Maestre, J; Tchénio, T; Dhellin, O; Heidmann, T

    1995-01-01

    Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome. Images PMID:8557053

  3. 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

  4. Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

    PubMed Central

    Fellous, A; Ginzburg, I; Littauer, U Z

    1982-01-01

    Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6964957

  5. 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

  6. qPCR based mRNA quality score show intact mRNA after heat stabilization

    PubMed Central

    Karlsson, Oskar; Segerström, Lova; Sjöback, Robert; Nylander, Ingrid; Borén, Mats

    2016-01-01

    Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality. PMID:27077049

  7. qPCR based mRNA quality score show intact mRNA after heat stabilization.

    PubMed

    Karlsson, Oskar; Segerström, Lova; Sjöback, Robert; Nylander, Ingrid; Borén, Mats

    2016-03-01

    Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality. PMID:27077049

  8. 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

  9. Dimethyl sulphoxide and haemin induce ferrochelatase mRNA by different mechanisms in murine erythroleukaemia cells.

    PubMed

    Fukuda, Y; Fujita, H; Taketani, S; Sassa, S

    1993-03-01

    The level of mRNA encoding ferrochelatase (FeC), the terminal enzyme of the haem biosynthetic pathway, was examined in murine erythroleukaemia (MEL) cells when they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulphoxide (DMSO), or haemin. FeC mRNA increased within 12 h after DMSO or haemin treatment of MEL cells, and its level continued to increase for 48 h. Treatment of cells with succinylacetone (SA), a potent inhibitor of haem synthesis, suppressed a DMSO-mediated increase in FeC mRNA, and haemin treatment reversed a SA-mediated decrease in FeC mRNA. Nuclear runoff analyses showed that, while DMSO increased the rate of transcription of FeC mRNA, haemin did not. These results indicate that the induction of FeC mRNA by DMSO is largely transcriptional, while that by haemin is post-transcriptional. PMID:8485055

  10. 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

  11. Interleukin-2 (IL-2, Proleukin) and immune function.

    PubMed

    2003-01-01

    IL-2 is an immune-based therapy that results in dramatic increases in CD4+ cell counts when used in conjunction with anti-HIV therapy. Although IL-2 has been discussed in previous issues of PI Perspective, new information warrants a further look at the product. PMID:12647677

  12. 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)

  13. 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

  14. PABPN1-Dependent mRNA Processing Induces Muscle Wasting

    PubMed Central

    Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

    2016-01-01

    Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

  15. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    PubMed

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. PMID:26344770

  16. PABPN1-Dependent mRNA Processing Induces Muscle Wasting.

    PubMed

    Riaz, Muhammad; Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D; Florea, Bogdan I; Raz, Vered

    2016-05-01

    Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

  17. Serum Stable Natural Peptides Designed by mRNA Display

    PubMed Central

    Howell, Shannon M.; Fiacco, Stephen V.; Takahashi, Terry T.; Jalali-Yazdi, Farzad; Millward, Steven W.; Hu, Biliang; Wang, Pin; Roberts, Richard W.

    2014-01-01

    Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence. PMID:25234472

  18. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  19. 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.

  20. alpha 1-Fetoprotein mRNA of rat yolk sac and hepatoma.

    PubMed Central

    Chiu, J F; Decha-Umphai, W; Commer, P

    1979-01-01

    Rat alpha 1-fetoprotein mRNA was isolated and purified to apparent homogeneity by means of immunoadsorption and oligo (dT) cellulose affinity chromatography. Purified AFP mRNA migrated as a 21S peak in 2.5% SDS-polyacrylamide gels. The translation product of this mRNA in micrococcal nuclease treated reticulocyte lysate was identified as AFP by specific immunoprecipitation, SDS-gel electrophoresis and tryptic digestion analysis. DNA complimentary to AFP mRNA was synthesized with avian meyloblastosis virus RNA-dependent DNA polymerase. This AFP cDNA was used as a probe to quantitate AFP mRNA in the developing rat liver and to compare the complexity and diversity of AFP mRNA derived from the normal rat liver and Morris hepatoma 7777. We found that the amount of functional AFP mRNA is decreasing during liver development. There is very little, if any, AFP mRNA in the adult rat liver. A high degree of homology between the AFP mRNA sequences of yolk sac and hepatoma was also found. PMID:91159

  1. Regulation of neuronal oxytocin mRNA by ovarian steroids in the mature and developing hypothalamus.

    PubMed Central

    Miller, F D; Ozimek, G; Milner, R J; Bloom, F E

    1989-01-01

    We have examined the changes in neuronal expression of oxytocin mRNA in the perinatal and mature female rat as a function of endogenous gonadal steroids. Northern blot analysis demonstrated a significant developmental increase in the abundance of oxytocin mRNA in the female brain concomitant with puberty. Ovariectomy of adult females decreased total brain oxytocin mRNA to significantly lower levels. In contrast, lactating mothers had increased levels of neuronal oxytocin mRNA. In situ hybridization analysis of neuronal oxytocin mRNA in adolescent, mature virgin, and ovariectomized virgin female brains demonstrated that the location and number of neurons expressing oxytocin mRNA was unchanged and that total brain oxytocin mRNA differences were attributable to amounts expressed per neuron. Differences in mRNA abundance were noted in oxytocin neurons throughout the hypothalamus, including those known to project as magnocellular neurons to the neurohypophysis and those of parvocellular origin thought to make wholly intracerebral connections. This developmental and dynamic regulation of oxytocin mRNA levels during gonadal maturation may coordinate the peripheral and central effects of this peptide on the reproductive biology of the female rat. Images PMID:2928343

  2. Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

    PubMed Central

    Gong, Z Y; Brandhorst, B P

    1988-01-01

    An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed. Images PMID:3211150

  3. All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay.

    PubMed

    Belasco, Joel G

    2010-07-01

    Despite its universal importance for controlling gene expression, mRNA degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. This conclusion was based on differences in the structures used by these organisms to protect mRNA termini and in the RNases and modifying enzymes originally implicated in mRNA decay. Subsequent discoveries have identified several striking parallels between the cellular factors and molecular events that govern mRNA degradation in these two kingdoms of life. Nevertheless, some key distinctions remain, the most fundamental of which may be related to the different mechanisms by which eukaryotes and bacteria control translation initiation. PMID:20520623

  4. Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor mRNA binding protein in human mesothelioma cells.

    PubMed Central

    Shetty, S; Kumar, A; Idell, S

    1997-01-01

    Treatment of human pleural mesothelioma (MS-1) cells with phorbol myristate acetate (PMA) and cycloheximide results in 17- and 10-fold, respectively, increases in steady-state expression of urokinase-type plasminogen activator receptor (uPAR) mRNA. Studies of transcriptional inhibition by actinomycin D showed four- and sixfold extensions of uPAR mRNA half-life in MS-1 cells treated with PMA and cycloheximide, respectively, suggesting that uPAR gene expression involves a posttranscriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 50-kDa uPAR mRNA binding protein (uPAR mRNABp) that selectively bound to a 51-nucleotide (nt) fragment of mRNA corresponding to the uPAR coding region. We investigated the possibility that this 51-nt protein binding fragment of uPAR mRNA contains regulatory information for message stability. Chimeric beta-globin/uPAR/beta-globin mRNA containing the 51-nt protein binding fragment was able to destabilize otherwise stable beta-globin mRNA. Conversely, a control chimeric beta-globin/uPAR/beta-globin mRNA containing a 51-nt fragment of the uPAR coding region that does not bind uPAR mRNABp was stable under identical conditions. Binding of uPAR mRNABp to uPAR mRNA was abolished after treatment with cycloheximide and rapidly down-regulated by PMA. These data suggest that the 51-nt protein binding fragment of uPAR mRNA may be involved in mRNA turnover as well as in cycloheximide-induced uPAR message stabilization. Our results indicate a novel mechanism of uPAR gene regulation in which cis elements within a 51-nt coding region interact with a uPAR mRNABp to regulate uPAR message stability. PMID:9032234

  5. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-β -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 β -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and β -globin sequences, we show that the NS1 5' splice site was effectively utilized by the β -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 β -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from β -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.

  6. Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

    PubMed

    Ernfors, Patrik; Persson, Håkan

    1991-01-01

    Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia. PMID:12106253

  7. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    PubMed

    Blair, E D; Blair, C C; Wagner, E K

    1987-08-01

    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. PMID:3037112

  8. Interleukin-2/Anti-Interleukin-2 Immune Complex Attenuates Cardiac Remodeling after Myocardial Infarction through Expansion of Regulatory T Cells

    PubMed Central

    Zeng, Zhipeng; Yu, Kunwu; Chen, Long; Li, Weihua; Xiao, Hong; Huang, Zhengrong

    2016-01-01

    CD4+CD25+Foxp3+ regulatory T cells (Treg cells) have protective effects in wound healing and adverse ventricular remodeling after myocardial infarction (MI). We hypothesize that the interleukin- (IL-) 2 complex comprising the recombinant mouse IL-2/anti-IL-2 mAb (JES6-1) attenuates cardiac remodeling after MI through the expansion of Treg. Mice were subjected to surgical left anterior descending coronary artery ligation and treated with either PBS or IL-2 complex. The IL-2 complex significantly attenuates ventricular remodeling, as demonstrated by reduced infarct size, improved left ventricular (LV) function, and attenuated cardiomyocyte apoptosis. The IL-2 complex increased the percentage of CD4+CD25+Foxp3+ Treg cells, which may be recruited to the infarcted heart, and decreased the frequencies of IFN-γ- and IL-17-producing CD4+ T helper (Th) cells among the CD4+Foxp3− T cells in the spleen. Furthermore, the IL-2 complex inhibited the gene expression of proinflammatory cytokines as well as macrophage infiltrates in the infarcted myocardium and induced the differentiation of macrophages from M1 to M2 phenotype in border zone of infarcted myocardium. Our studies indicate that the IL-2 complex may serve as a promising therapeutic approach to attenuate adverse remodeling after MI through expanding Treg cells specifically. PMID:27144181

  9. 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

  10. Genome-wide analysis of mRNA decay patterns during early Drosophila development

    PubMed Central

    2010-01-01

    Background The modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development. Results Here we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo. Conclusions Our work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay. PMID:20858238

  11. Regulation of the mRNA half-life in breast cancer.

    PubMed

    Griseri, Paola; Pagès, Gilles

    2014-08-10

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3'UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  12. Regulation of the mRNA half-life in breast cancer

    PubMed Central

    Griseri, Paola; Pagès, Gilles

    2014-01-01

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3’UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  13. 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...

  14. 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

  15. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    PubMed

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    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

  16. Nucleolin links to arsenic-induced stabilization of GADD45alpha mRNA.

    PubMed

    Zhang, Yadong; Bhatia, Deepak; Xia, Hongfeng; Castranova, Vince; Shi, Xianglin; Chen, Fei

    2006-01-01

    The present study shows that arsenic induces GADD45alpha (growth arrest and DNA damage inducible gene 45alpha) mainly through post-transcriptional mechanism. Treatment of the human bronchial epithelial cell line, BEAS-2B, with arsenic(III) chloride (As3+) resulted in a significant increase in GADD45alpha protein and mRNA. However, As3+ only exhibited a marginal effect on the transcription of the GADD45alpha gene. The accumulation of GADD45alpha mRNA is largely achieved by the stabilization of GADD45alpha mRNA in the cellular response to As3+. As3+ is able to induce binding of mRNA stabilizing proteins, nucleolin and less potently, HuR, to the GADD45alpha mRNA. Although As3+ was unable to affect the expression of nucleolin, treatment of the cells with As3+ resulted in re-distribution of nucleolin from nucleoli to nucleoplasm. Silencing of the nucleolin mRNA by RNA interference reversed As3+-induced stabilization of the GADD45alpha mRNA and accumulation of the GADD45alpha protein. Stabilization of GADD45alpha mRNA, thus, represents a novel mechanism contributing to the production of GADD45alpha and cell cycle arrest in response to As3+. PMID:16421274

  17. Nucleolin links to arsenic-induced stabilization of GADD45α mRNA

    PubMed Central

    Zhang, Yadong; Bhatia, Deepak; Xia, Hongfeng; Castranova, Vince; Shi, Xianglin; Chen, Fei

    2006-01-01

    The present study shows that arsenic induces GADD45α (growth arrest and DNA damage inducible gene 45α) mainly through post-transcriptional mechanism. Treatment of the human bronchial epithelial cell line, BEAS-2B, with arsenic(III) chloride (As3+) resulted in a significant increase in GADD45α protein and mRNA. However, As3+ only exhibited a marginal effect on the transcription of the GADD45α gene. The accumulation of GADD45α mRNA is largely achieved by the stabilization of GADD45α mRNA in the cellular response to As3+. As3+ is able to induce binding of mRNA stabilizing proteins, nucleolin and less potently, HuR, to the GADD45α mRNA. Although As3+ was unable to affect the expression of nucleolin, treatment of the cells with As3+ resulted in re-distribution of nucleolin from nucleoli to nucleoplasm. Silencing of the nucleolin mRNA by RNA interference reversed As3+-induced stabilization of the GADD45α mRNA and accumulation of the GADD45α protein. Stabilization of GADD45α mRNA, thus, represents a novel mechanism contributing to the production of GADD45α and cell cycle arrest in response to As3+. PMID:16421274

  18. 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

  19. 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

  20. 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...

  1. Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine.

    PubMed

    Popp, Maximilian W; Maquat, Lynne E

    2016-06-01

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA quality control and regulatory process that plays direct roles in human health and disease. In this Minireview, we discuss how understanding the molecular events that trigger NMD can facilitate strategic targeting of genes via CRISPR/Cas9 technologies and also inform disease diagnostics and treatments. PMID:27259145

  2. Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA.

    PubMed

    Díaz-Toledano, Rosa; Gómez, 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 200 nts, 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

  3. Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product

    PubMed Central

    Braun, Katherine A.; Dombek, Kenneth M.

    2015-01-01

    In the yeast Saccharomyces cerevisiae, the switch from respiratory metabolism to fermentation causes rapid decay of transcripts encoding proteins uniquely required for aerobic metabolism. Snf1, the yeast ortholog of AMP-activated protein kinase, has been implicated in this process because inhibiting Snf1 mimics the addition of glucose. In this study, we show that the SNF1-dependent ADH2 promoter, or just the major transcription factor binding site, is sufficient to confer glucose-induced mRNA decay upon heterologous transcripts. SNF1-independent expression from the ADH2 promoter prevented glucose-induced mRNA decay without altering the start site of transcription. SNF1-dependent transcripts are enriched for the binding motif of the RNA binding protein Vts1, an important mediator of mRNA decay and mRNA repression whose expression is correlated with decreased abundance of SNF1-dependent transcripts during the yeast metabolic cycle. However, deletion of VTS1 did not slow the rate of glucose-induced mRNA decay. ADH2 mRNA rapidly dissociated from polysomes after glucose repletion, and sequences bound by RNA binding proteins were enriched in the transcripts from repressed cells. Inhibiting the protein kinase A pathway did not affect glucose-induced decay of ADH2 mRNA. Our results suggest that Snf1 may influence mRNA stability by altering the recruitment activity of the transcription factor Adr1. PMID:26667037

  4. Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.

    PubMed

    Schmidt, Yvonne; Biniossek, Martin; Stark, G Björn; Finkenzeller, Günter; 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

  5. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    SciTech Connect

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  6. 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

  7. 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

  8. Regulation and deregulation of mRNA translation during myeloid maturation.

    PubMed

    Khanna-Gupta, Arati

    2011-02-01

    Gene expression in the eukaryotic cell is regulated at a number of levels, including transcription of genomic DNA into messenger RNA (mRNA), nucleocytoplasmic export of mRNA, and translation of the exported mRNA into proteins in the cytoplasm by ribosomes. The role played by epigenetics and transcription factors associated with the control of gene expression in the developing neutrophil has been well documented and appreciated over the years. A wealth of information on the role played by transcription factors in myeloid biology has contributed to our understanding of both normal and abnormal neutrophil development. However, regulation of mRNA translation in myeloid cell maturation is much less well-studied. A better understanding of the translational control of myeloid gene expression may provide important insights into both normal and abnormal myeloid maturation. This review summarizes our current understanding of the regulation of myeloid gene expression at the mRNA translational level. PMID:21093533

  9. 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

  10. Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

    PubMed

    Wada, E; Hisatomi, H; Moritoyo, T; Kanamaru, T; Hikiji, K

    1998-01-01

    This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis. PMID:9769378

  11. Visualization of dynamics of single endogenous mRNA labeled in live mouse.

    PubMed

    Park, Hye Yoon; Lim, Hyungsik; Yoon, Young J; Follenzi, Antonia; Nwokafor, Chiso; Lopez-Jones, Melissa; Meng, Xiuhua; Singer, Robert H

    2014-01-24

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment. PMID:24458643

  12. Cigarette Smoking and Tryptophan Hydroxylase 2 mRNA in the Dorsal Raphe Nucleus in Suicides

    PubMed Central

    Bach, Helene; Arango, Victoria; Kassir, Suham A.; Dwork, Andrew J.; Mann, J. John; Underwood, Mark D.

    2016-01-01

    Cigarette smoking is associated with suicide and mood disorders and stimulates serotonin release. Tryptophan hydroxylase (TPH2) synthesizes serotonin and is over-expressed in suicides. We determined whether smoking is associated with TPH2 mRNA in suicides and controls. TPH2 mRNA was measured postmortem in the dorsal raphe nucleus (DRN) of controls (N=26, 17 nonsmokers and nine smokers) and suicides (N=23, 5 nonsmokers and 18 smokers). Psychiatric history was obtained by psychological autopsy. TPH2 mRNA was greater in suicide nonsmokers than suicide smokers, control smokers and control nonsmokers (p=0.006). There was more TPH2 mRNA throughout the DRN. Smoking interferes with the TPH2 mRNA increase observed in suicide nonsmokers. The absence of altered TPH2 expression in non-suicide smokers suggests no pharmacological effect of smoking. PMID:26954509

  13. 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

  14. 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

  15. 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.

  16. PURE mRNA display for in vitro selection of single-chain antibodies.

    PubMed

    Nagumo, Yu; Fujiwara, Kei; Horisawa, Kenichi; Yanagawa, Hiroshi; Doi, Nobuhide

    2016-05-01

    mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications. PMID:26711234

  17. T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats.

    PubMed

    Silva, F Goulart da; Giannocco, G; Luchessi, A D; Curi, R; Nunes, M T

    2010-04-12

    Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-I mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. PMID:20015464

  18. Intranasal mRNA nanoparticle vaccination induces prophylactic and therapeutic anti-tumor immunity

    PubMed Central

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

    2014-01-01

    Direct in vivo administration of messenger RNA (mRNA) delivered in both naked and nanoparticle formats are actively investigated because the use of dendritic cells transfected ex vivo with mRNA for cancer therapy is expensive and needs significant infrastructure. Notably, intravenous and subcutaneous injections are the only routes of administration tested for mRNA nanoparticle tumor vaccination. In this report, we demonstrate that tumor immunity can be achieved via nasal administration of mRNA. Mice nasally immunized with mRNA delivered in nanoparticle format demonstrate delayed tumor progression in both prophylactic and therapeutic immunization models. The observed tumor immunity correlates with splenic antigen-specific CD8+ T cells and is achieved only when mRNA is delivered in nanoparticle but not in naked format. In conclusion, we demonstrate, as a proof-of-concept, a non-invasive approach to mRNA tumor vaccination, increasing its potential as a broadly applicable and off-the-shelf therapy for cancer treatment. PMID:24894817

  19. Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.

    PubMed Central

    Kinniburgh, A J; Martin, T E

    1976-01-01

    RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA. PMID:1066686

  20. 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 TASEP—both 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.

  1. Elevated TREM2 mRNA expression in leukocytes in schizophrenia but not major depressive disorder.

    PubMed

    Yoshino, Yuta; Kawabe, Kentaro; Yamazaki, Kiyohiro; Watanabe, Shinya; Numata, Shusuke; Mori, Yoko; Yoshida, Taku; Iga, Junichi; Ohmori, Tetsuro; Ueno, Shu-Ichi

    2016-06-01

    The pathological mechanisms of schizophrenia (SCZ) have not been clarified, but the microglia hypothesis has recently been discussed. We previously reported that the mRNA for a protein related to activation of microglia, triggering receptor expressed on myeloid cell 2 (TREM2), is expressed higher in peripheral leukocytes in SCZ than controls. In this study, we analyzed TREM2 mRNA expression in leukocytes from both SCZ and major depressive disorder (MDD) patients. We compared 50 SCZ patients and 42 MDD patients with age-matched controls. Levels of TREM2 mRNA in leukocytes were analyzed with quantitative real-time PCR method using TaqMan probe. TREM2 mRNA expression was significantly higher in leukocytes of SCZ subjects than controls, but the expression level was non-significantly different in MDD subjects. We observed a decrease in TREM2 mRNA expression in leukocytes from one SCZ patient after clozapine treatment. The expression did not change following ECT, but the expression level in this patient was still significantly higher than that in controls. We conclude that the high amount of TREM2 mRNA expression in leukocytes is specific to SCZ but not MDD and that changes in TREM2 mRNA expression may be a trait biomarker for SCZ. PMID:27130565

  2. 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

  3. 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

  4. 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.

  5. Membrane-association of mRNA decapping factors is independent of stress in budding yeast

    PubMed Central

    Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

    2016-01-01

    Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

  6. Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

    PubMed

    Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

    2016-01-01

    Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

  7. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    PubMed

    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

  8. 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

  9. Prefrontal cortical-striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity.

    PubMed

    Simon, Nicholas W; Beas, Blanca S; Montgomery, Karienn S; Haberman, Rebecca P; Bizon, Jennifer L; Setlow, Barry

    2013-06-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  10. Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity

    PubMed Central

    Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

    2014-01-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  11. Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability

    PubMed Central

    Sellers, R S; Luchin, A I; Richard, V; Brena, R M; Lima, D; Rosol, T J

    2011-01-01

    Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3′-untranslated region (3′-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-β1 (TGF-β1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-β1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-β1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein–RNA binding studies identified different proteins binding to the 3′-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-β1-induced changes in PTHrP mRNA isoform expression and stability. PMID:15291755

  12. The Formation of the Bicoid Morphogen Gradient Requires Protein Movement from Anteriorly Localized mRNA

    PubMed Central

    Kneeland, Thomas B.; Wieschaus, Eric F.; Gregor, Thomas

    2011-01-01

    The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics. Recent work has proposed that bicoid mRNA spatial distribution is sufficient to produce the observed protein gradient, minimizing the role of protein transport. Here, we adapt a novel method of fluorescent in situ hybridization to quantify the global spatio-temporal dynamics of bicoid mRNA particles. We determine that >90% of all bicoid mRNA is continuously present within the anterior 20% of the embryo. bicoid mRNA distribution along the body axis remains nearly unchanged despite dynamic mRNA translocation from the embryo core to the cortex. To evaluate the impact of mRNA distribution on protein gradient dynamics, we provide detailed quantitative measurements of nuclear Bicoid levels during the formation of the protein gradient. We find that gradient establishment begins 45 minutes after fertilization and that the gradient requires about 50 minutes to reach peak levels. In numerical simulations of gradient formation, we find that incorporating the actual bicoid mRNA distribution yields a closer prediction of the observed protein dynamics compared to modeling protein production from a point source at the anterior pole. We conclude that the spatial distribution of bicoid mRNA contributes to, but cannot account for, protein gradient formation, and therefore that protein movement, either active or passive, is required for gradient formation. PMID:21390295

  13. Isolation and Amplification of mRNA within a Simple Microfluidic Lab on a Chip

    PubMed Central

    Reinholt, Sarah J.; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J.

    2014-01-01

    The major modules for realizing molecular biological assays in a micro total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic messenger RNA (mRNA) within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid non-specific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 1015 fold and still result in successful on-chip re-amplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to bench-top devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes. PMID:24328414

  14. Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea.

    PubMed

    Gonen, L; Viterbo, A; Cantone, F; Staples, R C; Mayer, A M

    1996-05-01

    The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds. RNA was isolated from the cultures and probed with specific DNA probes for laccase. As an internal control, the RNA was probed for Botrytis beta-tubulin mRNA. From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase. This could account for the previously observed repression of laccase formation by cucurbitacins. PMID:8688171

  15. All-in-one detector of circulating mRNA based on a smartphone

    NASA Astrophysics Data System (ADS)

    Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

    2016-03-01

    Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

  16. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana.

    PubMed

    Calderwood, Alexander; Kopriva, Stanislav; Morris, Richard J

    2016-03-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements. PMID:26952566

  17. Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR

    PubMed Central

    Ko, Gwangpyo; Cromeans, Theresa L.; Sobsey, Mark D.

    2003-01-01

    We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

  18. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana[OPEN

    PubMed Central

    Calderwood, Alexander

    2016-01-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements. PMID:26952566

  19. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    PubMed

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. PMID:26119729

  20. Localization of insulin receptor mRNA in rat brain by in situ hybridization

    SciTech Connect

    Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G. )

    1990-12-01

    Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three {sup 35}S-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.

  1. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. )

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  2. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    PubMed

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-01-01

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol. PMID:27323091

  3. Changed in translation: mRNA recoding by -1 programmed ribosomal frameshifting.

    PubMed

    Caliskan, Neva; Peske, Frank; Rodnina, Marina V

    2015-05-01

    Programmed -1 ribosomal frameshifting (-1PRF) is an mRNA recoding event commonly utilized by viruses and bacteria to increase the information content of their genomes. Recent results have implicated -1PRF in quality control of mRNA and DNA stability in eukaryotes. Biophysical experiments demonstrated that the ribosome changes the reading frame while attempting to move over a slippery sequence of the mRNA--when a roadblock formed by a folded downstream segment in the mRNA stalls the ribosome in a metastable conformational state. The efficiency of -1PRF is modulated not only by cis-regulatory elements in the mRNA but also by trans-acting factors such as proteins, miRNAs, and antibiotics. These recent results suggest a molecular mechanism and new important cellular roles for -1PRF. PMID:25850333

  4. Biological activity of mRNA immobilized on nitrocellulose in NaI.

    PubMed Central

    Bresser, J; Hubbell, H R; Gillespie, D

    1983-01-01

    In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells. Images PMID:6579539

  5. 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

  6. Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

    PubMed Central

    Hardin, P E; Hall, J C; Rosbash, M

    1992-01-01

    The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator. Images PMID:1465387

  7. Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway.

    PubMed

    Czyzyk-Krzeska, M F; Lawson, E E; Millhorn, D E

    1992-11-01

    Dopamine is a major neurotransmitter in the arterial chemoreceptor pathway. In the present study we wished to determine if messenger RNAs for dopamine D1 and D2 receptor are expressed in carotid body (type I cells), in sensory neurons of the petrosal ganglion which innervate the carotid body and in sympathetic neurons of the superior cervical ganglion. We failed to detect D1 receptor mRNA in any of these tissues. However, we found that D2 receptor mRNA was expressed by dopaminergic carotid body type I cells. D2 receptor mRNA was also found in petrosal ganglion neurons that innervated the carotid sinus and carotid body. In addition, a large number of sympathetic postganglionic neurons in the superior cervical ganglion expressed D2 receptor mRNA. PMID:1362730

  8. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences.

    PubMed

    Rhoads, Robert E

    2016-01-01

    Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA. PMID:27236789

  9. 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.

  10. 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.

  11. FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

    EPA Science Inventory

    In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

  12. Synthesis and activity of a novel inhibitor of nonsense-mediated mRNA decay.

    PubMed

    Gotham, Victoria J B; Hobbs, Melanie C; Burgin, Ryan; Turton, David; Smythe, Carl; Coldham, Iain

    2016-01-27

    During efforts to prepare the known compound , a new tetracyclic compound, called , was prepared in six steps. This compound was found to have good activity as an inhibitor of nonsense-mediated mRNA decay. PMID:26740124

  13. Improved protocol for the extraction of bacterial mRNA from soils.

    PubMed

    Sharma, Shilpi; Mehta, Ravikumar; Gupta, Rashi; Schloter, Michael

    2012-10-01

    An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. PMID:22841738

  14. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

    PubMed

    Roundtree, Ian A; He, Chuan

    2016-06-01

    N(6)-Methyladenosine (m(6)A) is emerging as a chemical mark that broadly affects the flow of genetic information in various biological processes in eukaryotes. Recently, Xiao et al. reported that the nuclear m(6)A reader protein YTHDC1 impacts mRNA splicing, providing a transcriptome-wide glance of splicing changes affected by this mRNA methylation reader protein. PMID:27050931

  15. 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

  16. An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

    PubMed

    Nassar, Farah J; El Eit, Rabab; Nasr, Rihab

    2016-01-01

    Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis. PMID:27581151

  17. Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.

    PubMed

    Singhania, Richa; Pavey, Sandra; Payne, Elizabeth; Gu, Wenyi; Clancy, Jennifer; Jubair, Luqman; Preiss, Thomas; Saunders, Nicholas; McMillan, Nigel A J

    2016-08-01

    Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing. PMID:27321990

  18. Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin.

    PubMed

    Galbiati, Valentina; Carne, Alice; Mitjans, Montserrat; Galli, Corrado Lodovico; Marinovich, Marina; Corsini, Emanuela

    2012-02-01

    We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element-binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100 μg/ml (0.61 mM) for isoeugenol, 100 μg/ml (0.58 mM) for DEM, 3 μg/ml (14.8 μM) for DNCB, and 250 μg/ml (1.81 mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release. PMID:21969073

  19. Prognostic relevance of circulating CK19 mRNA in advanced malignant biliary tract diseases

    PubMed Central

    Leelawat, Kawin; Narong, Siriluck; Udomchaiprasertkul, Wandee; Wannaprasert, Jerasak; Treepongkaruna, Sa-ard; Subwongcharoen, Somboon; Ratanashu-ek, Tawee

    2012-01-01

    AIM: To determine the role of circulating tumor cells (CTCs) in prediction of the overall survival of patients with advanced malignant biliary tract obstruction. METHODS: We investigated the prognostic value of CTCs by examining two markers, cytokeratin (CK) 19 and human telomerase reverse transcriptase (hTERT) mRNA, in 40 patients diagnosed with advanced malignant biliary tract diseases. Quantitative real-time reverse transcription polymerase chain reaction was used to detect CK19 and hTERT mRNA in the peripheral blood of these patients. Overall survival was analyzed using the Kaplan-Meier method and Cox regression modeling. RESULTS: Positive CK19 and hTERT mRNA expression was detected in 45% and 60%, respectively, of the 40 patients. Univariable analysis indicated that positive CK19 mRNA expression was significantly associated with worse overall survival (P = 0.009). Multivariable analysis determined that positive CK19 mRNA expression, patient’s age and serum bilirubin were each independently associated with overall survival. CONCLUSION: CK19 mRNA expression levels in peripheral blood appear to provide a valuable marker to predict the overall survival of patients with advanced malignant biliary tract obstruction. PMID:22253524

  20. Differential Control of Interleukin-6 mRNA Levels by Cellular Distribution of YB-1

    PubMed Central

    Kang, Sujin; Lee, Taeyun A.; Ra, Eun A.; Lee, Eunhye; Choi, Hyun jin; Lee, Sungwook; Park, Boyoun

    2014-01-01

    Cytokine